Composite biofabricated material

ABSTRACT

The invention is directed to a composite material comprising a biofabricated material and a secondary component. The secondary component may be a porous material, such as a sheet of paper, cellulose, or fabric that has been coated or otherwise contacted with the biofabricated material. The biofabricated material comprises a uniform network of crosslinked collagen fibrils and provides strength, elasticity and an aesthetic appearance to the composite material.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/295,435 filed Feb. 15, 2016 and which is incorporated by reference inits entirety. This application is related to U.S. patent applicationSer. No. 13/853,001, titled “ENGINEERED LEATHER AND METHODS OFMANUFACTURE THEREOF” and filed on Mar. 28, 2013; U.S. patent applicationSer. No. 14/967,173, titled “ENGINEERED LEATHER AND METHODS OFMANUFACTURE THEREOF” and filed on Dec. 11, 2015; and PCT PatentApplication No. PCT/US2015/058794, titled “REINFORCED ENGINEEREDBIOMATERIALS AND METHODS OF MANUFACTURE THEREOF” and filed on Nov. 3,2015.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to biofabricated leather materials composed ofunbundled and randomly-oriented trimeric collagen fibrils that exhibitsuperior strength, non-anisotropic properties, and uniformity bycomparison to conventional leather products, but which have the look,feel and other aesthetic properties of natural leather. Unlike syntheticleather products composed of plastic resins, the biofabricated leatherof the invention is based on collagen, a natural component of leather.

Description of Related Art

Leather. Leather is used in a vast variety of applications, includingfurniture upholstery, clothing, shoes, luggage, handbag and accessories,and automotive applications. The estimated global trade value in leatheris approximately US $100 billion per year (Future Trends in the WorldLeather Products Industry and Trade, United Nations IndustrialDevelopment Organization, Vienna, 2010) and there is a continuing andincreasing demand for leather products. New ways to meet this demand arerequired in view of the economic, environmental and social costs ofproducing leather. To keep up with technological and aesthetic trends,producers and users of leather products seek new materials exhibitingsuperior strength, uniformity, processability and fashionable andappealing aesthetic properties that incorporate natural components.

Natural leathers are produced from the skins of animals which requireraising livestock. However, the raising of livestock requires enormousamounts of feed, pastureland, water, and fossil fuels. It also producesair and waterway pollution, including production of greenhouse gaseslike methane. Some states in the United States, such as California, mayimpose taxes on the amounts of pollutants such as methane produced bylivestock. As the costs of raising livestock rise, the cost of leatherwill rise.

The global leather industry slaughters more than a billion animals peryear. Most leather is produced in countries that engage in factoryfarming, lack animal welfare laws, or in which such laws go largely orcompletely unenforced. This slaughter under inhumane conditions isobjectionable to many socially conscious people. Consequently, there isa demand from consumers with ethical, moral or religious objections tothe use of natural leather products for products humanely producedwithout the mistreatment or slaughter of animals or produced in waysthat minimize the number of animals slaughtered.

The handling and processing of animal skins into leather also poseshealth risks because the handling animal skins can expose workers toanthrax and other pathogens and allergens such as those in leather dust.Factory farming of animals contributes to the spread of influenza (e.g.“bird flu”) and other infectious diseases that may eventually mutate andinfect humans. Animal derived products are also susceptible tocontamination with viruses and prions (“mad cow disease”). For producerand consumer peace of mind, there exists a demand for leather productsthat do not present these risks.

Natural leather is generally a durable and flexible material created byprocessing rawhide and skin of an animal, such as cattle hides. Thisprocessing typically involves three main parts: preparatory stages,tanning, and retanning. Leather may also be surface coated or embossed.

Numerous ways are known to prepare a skin or hide and convert it toleather. These include salting or refrigerating a hide or skin topreserve it; soaking or rehydrating the hide in an aqueous solution thatcontains surfactants or other chemicals to remove salt, dirt, debris,blood, and excess fat; defleshing or removing subcutaneous material fromthe hide; dehairing or unhairing the hide remove most of the hair;liming the hide to loosen fibers and open up collagen bundles allowingit to absorb chemicals; splitting the hide into two or more layers;deliming the hide to remove alkali and lower its pH; bating the hide tocomplete the deliming process and smooth the grain; degreasing to removeexcess fats; frizzing; bleaching; pickling by altering the pH; ordepickling,

Once the preparatory stages are complete, the leather is tanned. Leatheris tanned to increase its durability compared to untreated hide. Tanningconverts proteins in the hide or skin into a stable material that willnot putrefy while allowing the leather material to remain flexible.During tanning, the skin structure may be stabilized in an “open” formby reacting some of the collagen with complex ions of chromium or othertanning agents. Depending on the compounds used, the color and textureof the leather may change.

Tanning is generally understood to be the process of treating the skinsof animals to produce leather. Tanning may be performed in any number ofwell-understood ways, including by contacting a skin or hide with avegetable tanning agent, chromium compound, aldehyde, syntan, synthetic,semisynthetic or natural resin or polymer, or/and tanning natural oil ormodified oil. Vegetable tannins include pyrogallol- orpyrocatechin-based tannins, such as valonea, mimosa, ten, tara, oak,pinewood, sumach, quebracho and chestnut tannins; chromium tanningagents include chromium salts like chromium sulfate; aldehyde tanningagents include glutaraldehyde and oxazolidine compounds, syntans includearomatic polymers, polyacrylates, polymethacrylates, copolymers ofmaleic anhydride and styrene, condensation products of formaldehyde withmelamine or dicyandiamide, lignins and natural flours.

Chromium is the most commonly used tanning material. The pH of theskin/hide may be adjusted (e.g., lowered, e.g. to pH 2.8-3.2) to allowpenetration of the tanning agent; following penetration the pH may beraised to fix the tanning agent (“basification” to a slightly higherlevel, e.g., pH 3.8-4.2 for chrome).

After tanning, a leather may be retanned. Retanning refers to thepost-tanning treatment that can include coloring (dying), thinning,drying or hydrating, and the like. Examples of retanning techniquesinclude: tanning, wetting (rehydrating), sammying (drying),neutralization (adjusting pH to a less acidic or alkaline state),dyeing, fat liquoring, fixation of unbound chemicals, setting,conditioning, softening, buffing, etc.

A tanned leather product may be mechanically or chemically finished.Mechanical finishing can polish the leather to yield a shiny surface,iron and plate a leather to have a flat, smooth surface, emboss aleather to provide a three dimensional print or pattern, or tumble aleather to provide a more evident grain and smooth surface. Chemicalfinishing may involve the application of a film, a natural or syntheticcoating, or other leather treatment. These may be applied, for example,by spraying, curtain-coating or roller coating.

In animal hide, variations in fibrous collagen organization are observedin animals of different ages or species. These differences affect thephysical properties of hides and differences in leather produced fromthe hides. Variations in collagen organization also occur through thethickness of the hide. The top grain side of hide is composed of a finenetwork of collagen fibrils while deeper sections (corium) are composedof larger fiber bundles (FIG. 2). The smaller fibril organization of thegrain layer gives rise to a soft and smooth leather aesthetic while thelarger fiber bundle organization of deeper regions gives rise to a roughand course leather aesthetic. The porous, fibrous organization ofcollagen in a hide allows applied molecules to penetrate, stabilize, andlubricate it during leather tanning. The combination of the innatecollagen organization in hide and the modifications achieved throughtanning give rise to the desirable strength, drape and aestheticproperties of leather.

The top grain surface of leather is often regarded as the most desirabledue to its smoothness and soft texture. This leather grain contains ahighly porous network of organized collagen fibrils. Endogenous collagenfibrils are organized to have lacunar regions and overlapping regions;see the hierarchical organization of collagen depicted by FIG. 1. Thestrengths, microscale porosity, and density of fibrils in a top grainleather allow tanning or fatliquoring agents to penetrate it, thusstabilizing and lubricating the collagen fibrils, producing a soft,smooth and strong leather that people desire.

Leather hides can be split to obtain leather that is mostly top grain.The split hide can be further abraded to reduce the coarser grainedcorium on the split side, but there is always some residual corium andassociated rough appearance. In order to produce leather with smoothgrain on both sides, it is necessary to combine two pieces of grain,corium side facing corium side and either sew them together or laminatethem with adhesives with the smooth top grain sides facing outward.There is a demand for a leather product that has a smooth, topgrain-like surface on both its sides, because this would avoid the needfor splitting, and sewing or laminating two split leather piecestogether.

Control of the final properties of leather is limited by the naturalvariation in collagen structure between different animal hides. Forexample, the relative thickness of grain to corium in goat hide issignificantly higher than that in kangaroo hide. In addition, the weaveangle of collagen fiber bundles in kangaroo corium are much moreparallel to the surface of the hide, while fiber bundles in bovinecorium are oriented in both parallel and perpendicular orientations tothe surface of the hide. Further, the density of fiber bundles varieswithin each hide depending on their anatomical location. Hide taken frombutt, belly, shoulder, and neck can have different compositions andproperties. The age of an animal also affects the composition of itshide, for example, juvenile bovine hide contains smaller diameter fibersthan the larger fiber bundles found in adult bovine hide.

The final properties of leather can be controlled to some extent throughthe incorporation of stabilizing and lubricating molecules into the hideor skin during tanning and returning, however, the selection of thesemolecules is limited by the need to penetrate the dense structure of theskin or hide. Particles as large as several microns in diameter havebeen incorporated into leather for enhanced lubrication; however,application of these particles is limited to hides with the largest poresizes, uniformly distributing the particles throughout the hide presentsmany challenges.

Due to the size limitations of materials that can uniformly penetratethe hide, leather composite materials are often laminates of leather andthin layers of other materials such as Kevlar or nylon for mechanicalreinforcement, or polyurethanes and acrylics for aesthetically desirablesurfaces. Construction of leather with a dispersed secondary materialphase has not been achieved.

To address this limitation of natural leather, the inventors describethe fabrication of leather composites in which a continuous phase ofcollagen fibrils can encapsulate dispersed fibers and three dimensionalmaterials. This technology enables the fabrication of a new class ofleather materials with enhanced functionality.

While fibrillation of soluble collagens and collagen-like proteins hasbeen widely explored to produce collagen hydrogels for biomedicalapplications, harnessing this phenomena to fabricate leather-likecomposite materials has never been reported. By starting with an aqueousmixture of collagen monomers or fibrils, virtually any material can bereadily added to the mixture and further encapsulated into biofabricatedleather. Further, the combination of a continuous collagen fibril phasewith encapsulated fiber phase, composite materials with a grain-likeaesthetic and a range of enhanced mechanical properties can be achieved.

Many leather applications require a durable product that doesn't rip ortear, even when the leather has been stitched together. Typical productsthat include stitched leather and require durable leather includeautomobile steering wheel covers, automobile seats, furniture, sportinggoods, sport shoes, sneakers, watch straps and the like. There is a needto increase the durability of biofabricated leather to improveperformance in these products.

The top grain surface of leather is often regarded as the most desirabledue to its soft texture and smooth surface. As discussed previously, thegrain is a highly porous network of collagen fibrils. The strength ofthe collagen fibril, microscale porosity, and density of fibrils in thegrain allow tanning agent penetration to stabilize and lubricate thefibrils, producing a soft, smooth and stable material that peopledesire. While the aesthetic of the grain is very desirable, the strengthand tear resistance of the grain is often a limitation for practicalapplication of the grain alone. Therefore, the grain is often backedwith corium, its naturally reinforcing collagen layer, or can be backedartificially with laminar layers of synthetic materials. The reinforcedcollagen composites described herein allow for a thick and uniformgrain-like material with tunable mechanical properties through controlof the continuous and dispersed phases.

In addition to enhanced mechanical properties, this bottom-upfabrication approach can also enable the encapsulation of materials foraesthetic functionality. For example, photoluminescent materials can beencapsulated into biofabricated leather. In traditional tanning, smallernanoparticles to single molecules such as dyes are used to produceuniform coloration and aesthetic in leather. Since incorporation of dyesand aesthetic features relies on penetration of these molecules into thehide or skin, patterned features with controlled spatial organizationshave not been possible with leather. Patterned photoluminescencefeatures would provide unique functionality to leather including brandlogos, personalization, aesthetically pleasing patterns, andanti-counterfeit technology.

The materials described herein can be used to produce biofabricatedleathers with patterned photoluminescence features. Methods for forminga network of collagen fibrils in the presence or around a patternedsubstrate allows the encapsulation of precisely controlled patterns withlarger dimensions within the biofabricated leather structure. Virtuallyany photoluminescent material can be incorporated or encapsulated in abiofabricated leather. In order to visualize the pattern, the lightemitted from the embedded photoluminescent molecule must penetratethrough the thickness of the leather. Recent studies have shown thatlight penetration into collagen rich materials such as skin is highlywavelength dependent and decreases exponentially through the thicknessof the material. Therefore, variables such as the emission wavelength ofthe embedded photoluminescent material and the distance of thephotoluminescent material from the surface of the biofabricated leatherneed to be considered to produce photoluminescent features that arevisible by eye. Likewise, the intensity of the embedded photoluminescentmaterial needs to be considered for features that are detectable byreaders other than the eye, such as light emitting scanners for example.Further, three dimensional objects can be encapsulated into thebiofabricated leather in order to produce unique surface textures andpatterns. Surface patterns of traditional leather materials are limitedby natural variations in the skin surface of the animal, or by theability to emboss patterns onto the grain surface of leather. In orderto achieve unique patterns with deep surface features, three dimensionalobjects can be embedded into biofabricated leather. These textures andpatterns provide unique aesthetic features and can be used as logos forbrand recognition.

Collagen. Collagen is a component of leather. Skin, or animal hide,contains significant amounts of collagen, a fibrous protein. Collagen isa generic term for a family of at least 28 distinct collagen types;animal skin is typically type I collagen, although other types ofcollagen can be used in forming leather including type III collagen.Collagens are characterized by a repeating triplet of amino acids,-(Gly-X—Y)_(n)— and approximately one-third of the amino acid residuesin collagen are glycine. X is often proline and Y is oftenhydroxyproline, though there may be up to 400 possible Gly-X—Y triplets.Different animals may produce different amino acid compositions of thecollagen, which may result in different properties and in differences inthe resulting leather.

The structure of collagen can consist of three intertwined peptidechains of differing lengths. Collagen triple helices (or monomers) maybe produced from alpha-chains of about 1,050 amino acids long, so thatthe triple helix takes the form of a rod of about approximately 300 nmlong, with a diameter of approximately 1.5 nm. In the production ofextracellular matrix by fibroblast skin cells, triple helix monomers maybe synthesized and the monomers may self-assemble into a fibrous form.These triple helices are held together by electrostatic interactionsincluding salt bridging, hydrogen bonding, Van der Waals interactions,dipole-dipole forces, polarization forces, hydrophobic interactions,and/or covalent bonding. Triple helices can be bound together in bundlescalled fibrils, and fibrils can further assemble to create fibers andfiber bundles (FIG. 1). Fibrils have a characteristic banded appearancedue to the staggered overlap of collagen monomers. The distance betweenbands is approximately 67 nm for Type I collagen. Fibrils and fiberstypically branch and interact with each other throughout a layer ofskin. Variations of the organization or crosslinking of fibrils andfibers may provide strength to the material. Fibers may have a range ofdiameters depending on the type of animal hide. In addition to type Icollagen, skin (hides) may include other types of collagen as well,including type III collagen (reticulin), type IV collagen, and type VIIcollagen.

Various types of collagen exist throughout the mammalian body. Forexample, besides being the main component of skin and animal hide, TypeI collagen also exists in cartilage, tendon, vascular ligature, organs,muscle, and the organic portion of bone. Successful efforts have beenmade to isolate collagen from various regions of the mammalian body inaddition to the animal skin or hide. Decades ago, researchers found thatat neutral pH, acid-solubilized collagen self-assembled into fibrilscomposed of the same cross-striated patterns observed in native tissue;Schmitt F. O. J. Cell. Comp Physiol. 1942; 20:11). This led to use ofcollagen in tissue engineering and a variety of biomedical applications.In more recent years, collagen has been harvested from bacteria andyeast using recombinant techniques.

Regardless of the type of collagen, all are formed and stabilizedthrough a combination of physical and chemical interactions includingelectrostatic interactions including salt bridging, hydrogen bonding,Van der Waals interactions, dipole-dipole forces, polarization forces,hydrophobic interactions, and covalent bonding often catalyzed byenzymatic reactions. For Type I collagen fibrils, fibers, and fiberbundles, its complex assembly is achieved in vivo during development andis critical in providing mechanical support to the tissue while allowingfor cellular motility and nutrient transport. Various distinct collagentypes have been identified in vertebrates. These include bovine, ovine,porcine, chicken, and human collagens.

Generally, the collagen types are numbered by Roman numerals, and thechains found in each collagen type are identified by Arabic numerals.Detailed descriptions of structure and biological functions of thevarious different types of naturally occurring collagens are availablein the art; see, e.g., Ayad et al. (1998) The Extracellular Matrix FactsBook, Academic Press, San Diego, Calif.; Burgeson, R E., and Nimmi(1992) “Collagen types: Molecular Structure and Tissue Distribution” inClin. Orthop. 282:250-272; Kielty, C. M. et al. (1993) “The CollagenFamily: Structure, Assembly And Organization In The ExtracellularMatrix,” Connective Tissue And Its Heritable Disorders, MolecularGenetics, And Medical Aspects, Royce, P. M. and B. Steinmann eds.,Wiley-Liss, NY, pp. 103-147; and Prockop, D. J- and K. I. Kivirikko(1995) “Collagens: Molecular Biology, Diseases, and Potentials forTherapy,” Annu. Rev. Biochem., 64:403-434.)

Type I collagen is the major fibrillar collagen of bone and skincomprising approximately 80-90% of an organism's total collagen. Type Icollagen is the major structural macromolecule present in theextracellular matrix of multicellular organisms and comprisesapproximately 20% of total protein mass. Type I collagen is aheterotrimeric molecule comprising two α1(I) chains and one α2(I) chain,encoded by the COL1A1 and COL1A2 genes, respectively. Other collagentypes are less abundant than type I collagen, and exhibit differentdistribution patterns. For example, type II collagen is the predominantcollagen in cartilage and vitreous humor, while type III collagen isfound at high levels in blood vessels and to a lesser extent in skin.

Type II collagen is a homotrimeric collagen comprising three identicalα1(II) chains encoded by the COL2A1 gene. Purified type II collagen maybe prepared from tissues by, methods known in the art, for example, byprocedures described in Miller and Rhodes (1982) Methods In Enzymology82:33-64.

Type III collagen is a major fibrillar collagen found in skin andvascular tissues. Type III collagen is a homotrimeric collagencomprising three identical α1(III) chains encoded by the COL3A1 gene.Methods for purifying type III collagen from tissues can be found in,for example, Byers et al. (1974) Biochemistry 13:5243-5248; and Millerand Rhodes, supra.

Type IV collagen is found in basement membranes in the form of sheetsrather than fibrils. Most commonly, type IV collagen contains two α1(IV)chains and one α2(IV) chain. The particular chains comprising type IVcollagen are tissue-specific. Type IV collagen may be purified using,for example, the procedures described in Furuto and Miller (1987)Methods in Enzymology, 144:41-61, Academic Press.

Type V collagen is a fibrillar collagen found in, primarily, bones,tendon, cornea, skin, and blood vessels. Type V collagen exists in bothhomotrimeric and heterotrimeric forms. One form of type V collagen is aheterotrimer of two a 1(V) chains and one α2(V) chain. Another form oftype V collagen is a heterotrimer of α1(V), α2(V), and α3(V) chains. Afurther form of type V collagen is a homotrimer of α1(V). Methods forisolating type V collagen from natural sources can be found, forexample, in Elstow and Weiss (1983) Collagen Rel. Res. 3:181-193, andAbedin et al. (1982) Biosci. Rep. 2:493-502.

Type VI collagen has a small triple helical region and two largenon-collagenous remainder portions. Type VI collagen is a heterotrimercomprising α1(VI), α2(VI), and α3(VI) chains. Type VI collagen is foundin many connective tissues. Descriptions of how to purify type VIcollagen from natural sources can be found, for example, in Wu et al.(1987) Biochem. J. 248:373-381, and Kielty et al. (1991) J. Cell Sci.99:797-807.

Type VII collagen is a fibrillar collagen found in particular epithelialtissues. Type VII collagen is a homotrimeric molecule of three α1(VII)chains. Descriptions of how to purify type VII collagen from tissue canbe found in, for example, Lunstrum et al. (1986) J. Biol. Chem.261:9042-9048, and Bentz et al. (1983) Proc. Natl. Acad. Sci. USA80:3168-3172.Type VIII collagen can be found in Descemet's membrane inthe cornea. Type VIII collagen is a heterotrimer comprising two α1(VIII)chains and one α2(VIII) chain, although other chain compositions havebeen reported. Methods for the purification of type VIII collagen fromnature can be found, for example, in Benya and Padilla (1986) J. Biol.Chem. 261:4160-4169, and Kapoor et al. (1986) Biochemistry 25:3930-3937.

Type IX collagen is a fibril-associated collagen found in cartilage andvitreous humor. Type IX collagen is a heterotrimeric molecule comprisingα1(IX), α2(IX), and α3 (IX) chains. Type IX collagen has been classifiedas a FACIT (Fibril Associated Collagens with Interrupted Triple Helices)collagen, possessing several triple helical domains separated bynon-triple helical domains. Procedures for purifying type IX collagencan be found, for example, in Duance, et al. (1984) Biochem. J.221:885-889; Ayad et al. (1989) Biochem. J. 262:753-761; and Grant etal. (1988) The Control of Tissue Damage, Glauert, A. M., ed., ElsevierScience Publishers, Amsterdam, pp. 3-28.

Type X collagen is a homotrimeric compound of α1(X) chains. Type Xcollagen has been isolated from, for example, hypertrophic cartilagefound in growth plates; See, e.g., Apte et al. (1992) Eur J Biochem 206(1):217-24.

Type XI collagen can be found in cartilaginous tissues associated withtype II and type IX collagens, and in other locations in the body. TypeXI collagen is a heterotrimeric molecule comprising α1(XI), α2(XI), andα3(XI) chains. Methods for purifying type XI collagen can be found, forexample, in Grant et al., supra.

Type XII collagen is a FACIT collagen found primarily in associationwith type I collagen. Type XII collagen is a homotrimeric moleculecomprising three α1(XII) chains. Methods for purifying type XII collagenand variants thereof can be found, for example, in Dublet et al. (1989)J. Biol. Chem. 264:13150-13156; Lunstrum et al. (1992) J. Biol. Chem.267:20087-20092; and Watt et al. (1992) J. Biol. Chem. 267:20093-20099.

Type XIII is a non-fibrillar collagen found, for example, in skin,intestine, bone, cartilage, and striated muscle. A detailed descriptionof type XIII collagen may be found, for example, in Juvonen et al.(1992) J. Biol. Chem. 267: 24700-24707.

Type XIV is a FACIT collagen characterized as a homotrimeric moleculecomprising α1(XIV) chains. Methods for isolating type XIV collagen canbe found, for example, in Aubert-Foucher et al. (1992) J. Biol. Chem.267:15759-15764,and Watt et al., supra.

Type XV collagen is homologous in structure to type XVIII collagen.Information about the structure and isolation of natural type XVcollagen can be found, for example, in Myers et al. (1992) Proc. Natl.Acad. Sci. USA 89:10144-10148; Huebner et al. (1992) Genomics14:220-224; Kivirikko et al. (1994) J. Biol. Chem. 269:4773-4779; andMuragaki, J. (1994) Biol. Chem. 264:4042-4046.

Type XVI collagen is a fibril-associated collagen, found, for example,in skin, lung fibroblast, and keratinocytes. Information on thestructure of type XVI collagen and the gene encoding type XVI collagencan be found, for example, in Pan et al. (1992) Proc. Natl. Acad. Sci.USA 89:6565-6569; and Yamaguchi et al. (1992) J. Biochem. 112:856-863.

Type XVII collagen is a hemidesmosal transmembrane collagen, also knownat the bullous pemphigoid antigen. Information on the structure of typeXVII collagen and the gene encoding type XVII collagen can be found, forexample, in Li et al. (1993) J. Biol. Chem. 268(12):8825-8834; andMcGrath et al. (1995) Nat. Genet. 11(1):83-86.

Type XVIII collagen is similar in structure to type XV collagen and canbe isolated from the liver. Descriptions of the structures and isolationof type XVIII collagen from natural sources can be found, for example,in Rehn and Pihlajaniemi (1994) Proc. Natl. Acad. Sci USA 91:4234-4238;Oh et al. (1994) Proc. Natl. Acad. Sci USA 91:4229-4233; Rehn et al.(1994) J. Biol. Chem. 269:13924-13935; and Oh et al. (1994) Genomics19:494-499.

Type XIX collagen is believed to be another member of the FACIT collagenfamily, and has been found in mRNA isolated from rhabdomyosarcoma cells.Descriptions of the structures and isolation of type XIX collagen can befound, for example, in Inoguchi et al. (1995) J. Biochem. 117:137-146;Yoshioka et al. (1992) Genomics 13:884-886; and Myers et al., J. Biol.Chem. 289:18549-18557 (1994).

Type XX collagen is a newly found member of the FACIT collagenousfamily, and has been identified in chick cornea. (See, e.g., Gordon etal. (1999) FASEB Journal 13:A1119; and Gordon et al. (1998), IOVS39:S1128.)

Any type of collagen, truncated collagen, unmodified orpost-translationally modified, or amino acid sequence-modified collagenthat can be fibrillated and crosslinked by the methods described hereincan be used to produce a biofabricated material or biofabricatedleather. Biofabricated leather may contain a substantially homogenouscollagen, such as only Type I or Type III collagen or may containmixtures of 2, 3, 4 or more different kinds of collagens.

Recombinant Collagen. Recombinant expression of collagen andcollagen-like proteins is known and is incorporated by reference toBell, EP 1232182B1, Bovine collagen and method for producing recombinantgelatin; Olsen, et al., U.S. Pat. No. 6,428,978, Methods for theproduction of gelatin and fill-length triple helical collagen inrecombinant cells; VanHeerde, et al., U.S. Pat. No. 8,188,230, Methodfor recombinant microorganism expression and isolation of collagen-likepolypeptides. Such recombinant collagens have not been used to produceleather.

Prokaryotic expression. In prokaryotic systems, such as bacterialsystems, a number of expression vectors may be advantageously selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities of the animal collagens and gelatins ofthe invention are to be produced, such as for the generation ofantibodies, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited to, the E. coli expression vectorpUR278 (Ruther et al. (1983) EMBO J. 2:1791), in which the codingsequence may be ligated into the vector in frame with the lac Z codingregion so that a hybrid AS-lacZ protein is produced; pIN vectors (Inouyeet al. (1985) Nucleic Acids Res. 13:3101-3109 and Van Heeke et al.(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors mayalso be used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned polypeptide of interestcan be released from the GST moiety. A recombinant collagen may comprisecollagen molecules that have not been post-translationally modified,e.g., not glycosylated or hydroxylated, or may comprise one or morepost-translational modifications, for example, modifications thatfacilitate fibrillation and formation of unbundled and randomly orientedfibrils of collagen molecules. A recombinant collagen molecule cancomprise a fragment of the amino acid sequence of a native collagenmolecule that can form trimeric collagen fibrils or a modified collagenmolecule or truncated collagen molecule having an amino acid sequence atleast 70, 80, 90, 95, 96, 97, 98, or 99% identical or similar to anative collagen amino acid sequence (or to a fibril forming regionthereof or to a segment substantially comprising [Gly-X—Y]_(n)), such asthose of bovine collagen, described by SEQ ID NOS: 1, 2 or 3 and byamino acid sequences of Col1A1, Col1A2, and Col1A3, described byAccession Nos. NP_001029211.1(https://_www.ncbi.nlm.nih.gov/protein/77404252, last accessed Feb. 9,2017), NP_776945.1 (https://_www.ncbi.nlm.nih.gov/protein/27806257 lastaccessed Feb. 9, 2017) and NP_001070299.1(https://_www.ncbi.nlm.nih.gov/protein/116003881 last accessed Feb. 9,2017) which are incorporated by reference. (These links have beeninactivated by inclusion of an underline after the double slash.)

Such recombinant or modified collagen molecules will generally comprisethe repeated -(Gly-X—Y)_(n)— sequence described herein.

BLASTN may be used to identify a polynucleotide sequence having at least70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, or 99% sequenceidentity to a reference polynucleotide such as a polynucleotide encodinga collagen polypeptide or encoding the amino acid sequences of SEQ IDNOS: 1, 2 or 3. A representative BLASTN setting optimized to find highlysimilar sequences uses an Expect Threshold of 10 and a Wordsize of 28,max matches in query range of 0, match/mismatch scores of 1/−2, andlinear gap cost. Low complexity regions may be filtered or masked.Default settings of a Standard Nucleotide BLAST are described by andincorporated by reference tohttps://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome(last accessed Jan. 27, 2017).

BLASTP can be used to identify an amino acid sequence having at least70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, or 99% sequenceidentity, or similarity to a reference amino acid, such as a collagenamino acid sequence, using a similarity matrix such as BLOSUM45,BLOSUM62 or BLOSUM80 where BLOSUM45 can be used for closely relatedsequences, BLOSUM62 for midrange sequences, and BLOSUM80 for moredistantly related sequences. Unless otherwise indicated a similarityscore will be based on use of BLOSUM62. When BLASTP is used, the percentsimilarity is based on the BLASTP positives score and the percentsequence identity is based on the BLASTP identities score. BLASTP“Identities” shows the number and fraction of total residues in the highscoring sequence pairs which are identical; and BLASTP “Positives” showsthe number and fraction of residues for which the alignment scores havepositive values and which are similar to each other. Amino acidsequences having these degrees of identity or similarity or anyintermediate degree of identity or similarity to the amino acidsequences disclosed herein are contemplated and encompassed by thisdisclosure. A representative BLASTP setting that uses an ExpectThreshold of 10, a Word Size of 3, BLOSUM 62 as a matrix, and GapPenalty of 11 (Existence) and 1 (Extension) and a conditionalcompositional score matrix adjustment. Other default settings for BLASTPare described by and incorporated by reference to the disclosureavailable at:https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome(last accessed Jan. 27, 2017).

Yeast expression. In one embodiment, collagen molecules are produced ina yeast expression system. In yeast, a number of vectors containingconstitutive or inducible promoters known in the art may be used;Ausubel et al., supra, Vol. 2, Chapter 13; Grant et al. (1987)Expression and Secretion Vectors for Yeast, in Methods in Enzymology,Ed. Wu & Grossman, Acad. Press, N.Y. 153:516-544; Glover (1986) DNACloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; Bitter (1987)Heterologous Gene Expression in Yeast, in Methods in Enzymology, Eds.Berger & Kimmel, Acad. Press, N.Y. 152:673-684; and The MolecularBiology of the Yeast Saccharomyces, Eds. Strathern et al., Cold SpringHarbor Press, Vols. I and II (1982).

Collagen can be expressed using host cells, for example, from the yeastSaccharomyces cerevisiae. This particular yeast can be used with any ofa large number of expression vectors. Commonly employed expressionvectors are shuttle vectors containing the 2P origin of replication forpropagation in yeast and the Col E1 origin for E. coli, for efficienttranscription of the foreign gene. A typical example of such vectorsbased on 2P plasmids is pWYG4, which has the 2P ORI-STB elements, theGAL1-10 promoter, and the 2P D gene terminator. In this vector, an Ncolcloning site is used to insert the gene for the polypeptide to beexpressed, and to provide the ATG start codon. Another expression vectoris pWYG7L, which has intact 2αORI, STB, REP1 and REP2, and the GAL1-10promoter, and uses the FLP terminator. In this vector, the encodingpolynucleotide is inserted in the polylinker with its 5′ ends at a BamHIor Ncol site. The vector containing the inserted polynucleotide istransformed into S. cerevisiae either after removal of the cell wall toproduce spheroplasts that take up DNA on treatment with calcium andpolyethylene glycol or by treatment of intact cells with lithium ions.

Alternatively, DNA can be introduced by electroporation. Transformantscan be selected, for example, using host yeast cells that areauxotrophic for leucine, tryptophan, uracil, or histidine together withselectable marker genes such as LEU2, TRP1, URA3, HIS3, or LEU2-D.

In one embodiment, polynucleotides encoding collagen are introduced intohost cells from the yeast Pichia. Species of non-Saccharomyces yeastsuch as Pichia pastoris appear to have special advantages in producinghigh yields of recombinant protein in scaled up procedures.Additionally, a Pichia expression kit is available from InvitrogenCorporation (San Diego, Calif.).

There are a number of methanol responsive genes in methylotrophic yeastssuch as Pichia pastoris, the expression of each being controlled bymethanol responsive regulatory regions, also referred to as promoters.Any of such methanol responsive promoters are suitable for use in thepractice of the present invention. Examples of specific regulatoryregions include the AOX1 promoter, the AOX2 promoter, thedihydroxyacetone synthase (DAS), the P40 promoter, and the promoter forthe catalase gene from P. pastoris, etc.

In other embodiments, the methylotrophic yeast Hansenula polymorpha isused. Growth on methanol results in the induction of key enzymes of themethanol metabolism, such as MOX (methanol oxidase), DAS(dihydroxyacetone synthase), and FMHD (formate dehydrogenase). Theseenzymes can constitute up to 30-40% of the total cell protein. The genesencoding MOX, DAS, and FMDH production are controlled by strongpromoters induced by growth on methanol and repressed by growth onglucose. Any or all three of these promoters may be used to obtainhigh-level expression of heterologous genes in H. polymorpha. Therefore,in one aspect, a polynucleotide encoding animal collagen or fragments orvariants thereof is cloned into an expression vector under the controlof an inducible H. polymorpha promoter. If secretion of the product isdesired, a polynucleotide encoding a signal sequence for secretion inyeast is fused in frame with the polynucleotide. In a furtherembodiment, the expression vector preferably contains an auxotrophicmarker gene, such as URA3 or LEU2, which may be used to complement thedeficiency of an auxotrophic host.

The expression vector is then used to transform H. polymorpha host cellsusing techniques known to those of skill in the art. A useful feature ofH. polymorpha transformation is the spontaneous integration of up to 100copies of the expression vector into the genome. In most cases, theintegrated polynucleotide forms multimers exhibiting a head-to-tailarrangement. The integrated foreign polynucleotide has been shown to bemitotically stable in several recombinant strains, even undernon-selective conditions. This phenomena of high copy integrationfurther ads to the high productivity potential of the system.

Fungal Expression. Filamentous fungi may also be used to produce thepresent polypeptides. Vectors for expressing and/or secretingrecombinant proteins in filamentous fungi are well known, and one ofskill in the art could use these vectors to express the recombinantanimal collagens of the present invention.

Plant Expression. In one aspect, an animal collagen is produced in aplant or plant cells. In cases where plant expression vectors are used,the expression of sequences encoding the collagens of the invention maybe driven by any of a number of promoters. For example, viral promoterssuch as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al. (1984)Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu etal. (1987) EMBO J. 6:307-311) may be used; alternatively, plantpromoters such as the small subunit of RUBISCO (Coruzzi et al. (1984)EMBO J. 3:1671-1680; Broglie et al. (1984) Science 224:838-843) or heatshock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al.(1986) Mol. Cell. Biol. 6:559-565) may be used. These constructs can beintroduced into plant cells by a variety of methods known to those ofskill in the art, such as by using Ti plasmids, Ri plasmids, plant virusvectors, direct DNA transformation, microinjection, electroporation,etc. For reviews of such techniques see, for example, Weissbach &Weissbach, Methods for Plant Molecular Biology, Academic Press, NY,Section VIII, pp. 421-463 (1988); Grierson & Corey, Plant MolecularBiology, 2d Ed., Blackie, London, Ch. 7-9 (1988); Transgenic Plants: AProduction System for Industrial and Pharmaceutical Proteins, Owen andPen eds., John Wiley & Sons, 1996; Transgenic Plants, Galun and Breimaneds, Imperial College Press, 1997; and Applied Plant Biotechnology,Chopra, Malik, and Bhat eds., Science Publishers, Inc., 1999.

Plant cells do not naturally produce sufficient amounts ofpost-translational enzymes to efficiently produce stable collagen.Therefore, where hydroxylation is desired, plant cells used to expressanimal collagens are supplemented with the necessary post-translationalenzymes to sufficiently produce stable collagen. In a preferredembodiment of the present invention, the post-translational enzyme isprolyl 4-hydroxylase.

Methods of producing the present animal collagens in plant systems maybe achieved by providing a biomass from plants or plant cells, whereinthe plants or plant cells comprise at least one coding sequence isoperably linked to a promoter to effect the expression of thepolypeptide, and the polypeptide is then extracted from the biomass.Alternatively, the polypeptide can be non-extracted, e.g., expressedinto the endosperm.

Plant expression vectors and reporter genes are generally known in theart; See, e.g., Gruber et al. (1993) in Methods of Plant MolecularBiology and Biotechnology, CRC Press. Typically, the expression vectorcomprises a nucleic acid construct generated, for example, recombinantlyor synthetically, and comprising a promoter that functions in a plantcell, wherein such promoter is operably linked to a nucleic acidsequence encoding an animal collagen or fragments or variants thereof,or a post-translational enzyme important to the biosynthesis ofcollagen.

Promoters drive the level of protein expression in plants. To produce adesired level of protein expression in plants, expression may be underthe direction of a plant promoter. Promoters suitable for use inaccordance with the present invention are generally available in theart; See, e.g., PCT Publication No. WO 91/19806. Examples of promotersthat may be used in accordance with the present invention includenon-constitutive promoters or constitutive promoters. These promotersinclude, but are not limited to, the promoter for the small subunit ofribulose-1,5-bis-phosphate carboxylase; promoters from tumor-inducingplasmids of Agrobacterium tumefaciens, such as the RUBISCO nopalinesynthase (NOS) and octopine synthase promoters; bacterial T-DNApromoters such as mas and ocs promoters; and viral promoters such as thecauliflower mosaic virus (CaMV) 19S and 35S promoters or the figwortmosaic virus 35S promoter.

The polynucleotide sequences of the present invention can be placedunder the transcriptional control of a constitutive promoter, directingexpression of the collagen or post-translational enzyme in most tissuesof a plant. In one embodiment, the polynucleotide sequence is under thecontrol of the cauliflower mosaic virus (CaMV) 35S promoter. The doublestranded caulimorvirus family has provided the single most importantpromoter expression for transgene expression in plants, in particular,the 35S promoter; See, e.g., Kay et al. (1987) Science 236:1299.Additional promoters from this family such as the figwort mosaic viruspromoter, etc., have been described in the art, and may also be used;See, e.g., Sanger et al. (1990) Plant Mol. Biol. 14:433-443; Medberry etal. (1992) Plant Cell 4:195-192; and Yin and Beachy (1995) Plant J.7:969-980.

The promoters used in polynucleotide constructs for expressing collagenmay be modified, if desired, to affect their control characteristics.For example, the CaMV promoter may be ligated to the portion of theRUBISCO gene that represses the expression of RUBISCO in the absence oflight, to create a promoter which is active in leaves, but not in roots.The resulting chimeric promoter may be used as described herein.

Constitutive plant promoters having general expression properties knownin the art may be used with the expression vectors of the presentinvention. These promoters are abundantly expressed in most planttissues and include, for example, the actin promoter and the ubiquitinpromoter; See, e.g., McElroy et al. (1990) Plant Cell 2:163-171; andChristensen et al. (1992) Plant Mol. Biol. 18:675-689.

Alternatively, the polypeptide of the present invention may be expressedin a specific tissue, cell type, or under more precise environmentalconditions or developmental control. Promoters directing expression inthese instances are known as inducible promoters. In the case where atissue-specific promoter is used, protein expression is particularlyhigh in the tissue from which extraction of the protein is desired.Depending on the desired tissue, expression may be targeted to theendosperm, aleurone layer, embryo (or its parts as scutellum andcotyledons), pericarp, stem, leaves tubers, roots, etc. Examples ofknown tissue-specific promoters include the tuber-directed class Ipatatin promoter, the promoters associated with potato tuber ADPGPPgenes, the soybean promoter of β-conglycinin (7S protein) which drivesseed-directed transcription, and seed-directed promoters from the zeingenes of maize endosperm; See, e.g., Bevan et al. (1986) Nucleic AcidsRes. 14: 4625-38; Muller et al. (1990) Mol. Gen. Genet. 224:136-46; Bray(1987) Planta 172: 364-370; and Pedersen et al. (1982) Cell 29:1015-26.

Collagen polypeptides can be produced in seed by way of seed-basedproduction techniques using, for example, canola, corn, soybeans, riceand barley seed. In such a process, for example, the product isrecovered during seed germination; See, e.g., PCT Publication Numbers WO9940210; WO 9916890; WO 9907206; U.S. Pat. No. 5,866,121; U.S. Pat. No.5,792,933; and all references cited therein. Promoters that may be usedto direct the expression of the polypeptides may be heterologous ornon-heterologous. These promoters can also be used to drive expressionof antisense nucleic acids to reduce, increase, or alter concentrationand composition of the present animal collagens in a desired tissue.

Other modifications that may be made to increase and/or maximizetranscription of the present polypeptides in a plant or plant cell arestandard and known to those in the art. For example a vector comprisinga polynucleotide sequence encoding a recombinant animal collagen, or afragment or variant thereof, operably linked to a promoter may furthercomprise at least one factor that modifies the transcription rate ofcollagen or related post-translational enzymes, including, but notlimited to, peptide export signal sequence, codon usage, introns,polyadenylation, and transcription termination sites. Methods ofmodifying constructs to increase expression levels in plants aregenerally known in the art; See, e.g. Rogers et al. (1985) J. Biol.Chem. 260:3731; and Cornejo et al. (1993) Plant Mol Biol 23:567-58. Inengineering a plant system that affects the rate of transcription of thepresent collagens and related post-translational enzymes, variousfactors known in the art, including regulatory sequences such aspositively or negatively acting sequences, enhancers and silencers, aswell as chromatin structure can affect the rate of transcription inplants, at least one of these factors may be utilized when expressing arecombinant animal collagen, including but not limited to the collagentypes described above.

The vectors comprising the present polynucleotides will typicallycomprise a marker gene which confers a selectable phenotype on plantcells. Usually, the selectable marker gene will encode antibioticresistance, with suitable genes including at least one set of genescoding for resistance to the antibiotic spectinomycin, the streptomycinphophotransferase (SPT) gene coding for streptomycin resistance, theneomycin phophotransferase (NPTH) gene encoding kanamycin or geneticinresistance, the hygromycin resistance, genes coding for resistance toherbicides which act to inhibit the action of acetolactate synthase(ALS), in particular, the sulfonylurea-type herbicides; e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance in particular the S4 and/or Hra mutations, genes coding forresistance to herbicides which act to inhibit action of glutaminesynthase, such as phophinothricin or basta; e.g. the bar gene, or othersimilar genes known in the art. The bar gene encodes resistance to theherbicide basta, the nptII gene encodes resistance to the antibioticskanamycin and geneticin, and the ALS gene encodes resistance to theherbicide chlorsulfuron.

Typical vectors useful for expression of foreign genes in plants arewell known in the art, including, but not limited to, vectors derivedfrom the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. Thesevectors are plant integrating vectors that upon transformation,integrate a portion of the DNA into the genome of the host plant; seee.g., Rogers et al. (1987) Meth In Enzymol. 153:253-277; Schardl et al.(1987) Gene 61:1-11; and Berger et al., Proc. Natl. Acad. Sci. U.S.A.86:8402-8406.

Vectors comprising sequences encoding the present polypeptides andvectors comprising post-translational enzymes or subunits thereof may beco-introduced into the desired plant. Procedures for transforming plantcells are available in the art, for example, direct gene transfer, invitro protoplast transformation, plant virus-mediated transformation,liposome-mediated transformation, microinjection, electroporation,Agrobacterium mediated transformation, and particle bombardment; seee.g., Paszkowski et al. (1984) EMBO J. 3:2717-2722; U.S. Pat. No.4,684,611; European Application No. 0 67 553; U.S. Pat. No. 4,407,956;U.S. Pat. No. 4,536,475; Crossway et al. (1986) Biotechniques 4:320-334;Riggs et al. (1986) Proc. Natl. Acad. Sci USA 83:5602-5606; Hinchee etal. (1988) Biotechnology 6:915-921; and U.S. Pat. No. 4,945,050.)Standard methods for the transformation of, e.g., rice, wheat, corn,sorghum, and barley are described in the art; See, e.g., Christou et al.(1992) Trends in Biotechnology 10: 239 and Lee et al. (1991) Proc. Nat'lAcad. Sci. USA 88:6389. Wheat can be transformed by techniques similarto those employed for transforming corn or rice. Furthermore, Casas etal. (1993) Proc. Nat'l Acad. Sci. USA 90:11212, describe a method fortransforming sorghum, while Wan et al. (1994) Plant Physiol. 104: 37,teach a method for transforming barley. Suitable methods for corntransformation are provided by Fromm et al. (1990) Bio/Technology 8:833and by Gordon-Kamm et al., supra.

Additional methods that may be used to generate plants that produceanimal collagens of the present invention are established in the art;See, e.g., U.S. Pat. No. 5,959,091; U.S. Pat. No. 5,859,347; U.S. Pat.No. 5,763,241; U.S. Pat. No. 5,659,122; U.S. Pat. No. 5,593,874; U.S.Pat. No. 5,495,071; U.S. Pat. No. 5,424,412; U.S. Pat. No. 5,362,865;U.S. Pat. No. 5,229,112; U.S. Pat. No. 5,981,841; U.S. Pat. No.5,959,179; U.S. Pat. No. 5,932,439; U.S. Pat. No. 5,869,720; U.S. Pat.No. 5,804,425; U.S. Pat. No. 5,763,245; U.S. Pat. No. 5,716,837; U.S.Pat. No. 5,689,052; U.S. Pat. No. 5,633,435; U.S. Pat. No. 5,631,152;U.S. Pat. No. 5,627,061; U.S. Pat. No. 5,602,321; U.S. Pat. No.5,589,612; U.S. Pat. No. 5,510,253; U.S. Pat. No. 5,503,999; U.S. Pat.No. 5,378,619; U.S. Pat. No. 5,349,124; U.S. Pat. No. 5,304,730; U.S.Pat. No. 5,185,253; U.S. Pat. No. 4,970,168; European Publication No.EPA 00709462; European Publication No. EPA 00578627; EuropeanPublication No. EPA 00531273; European Publication No. EPA 00426641; PCTPublication No. WO 99/31248; PCT Publication No. WO 98/58069; PCTPublication No. WO 9⁸/₄5457; PCT Publication No. WO 98/31812; PCTPublication No. WO 98/08962; PCT Publication No. WO 97/48814; PCTPublication No. WO 97/30582; and PCT Publication No. WO 9717459.

Insect Expression. Another alternative expression system for collagen isan insect system. Baculoviruses are very efficient expression vectorsfor the large scale production of various recombinant proteins in insectcells. The methods as described in Luckow et al. (1989) Virology170:31-39 and Gruenwald, S. and Heitz, J. (1993) Baculovirus ExpressionVector System: Procedures & Methods Manual, Pharmingen, San Diego,Calif., can be employed to construct expression vectors containing acollagen coding sequence for the collagens of the invention and theappropriate transcriptional/translational control signals. For example,recombinant production of proteins can be achieved in insect cells, byinfection of baculovirus vectors encoding the polypeptide. Theproduction of recombinant collagen, collagen-like or collagenouspolypeptides with stable triple helices can involve the co-infection ofinsect cells with three baculoviruses, one encoding the animal collagento be expressed and one each encoding the α subunit and β subunit ofprolyl 4-hydroxylase. This insect cell system allows for production ofrecombinant proteins in large quantities. In one such system, Autographacalifornica nuclear polyhidrosis virus (AcNPV) is used as a vector toexpress foreign genes. This virus grows in Spodoptera frugiperda cells.Coding sequences for collagen or collagen-like polypeptides may becloned into non-essential regions (for example the polyhedron gene) ofthe virus and placed under control of an AcNPV promoter (for example,the polyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus; e.g., viruses lacking the proteinaceouscoat coded for by the polyhedron gene. These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed; see, e.g., Smith et al. (1983) J. Virol. 46:584; andU.S. Pat. No. 4,215,051. Further examples of this expression system maybe found in, for example, Ausubel et al. above.

Animal Expression. In animal host cells, a number of expression systemsmay be utilized. In cases where an adenovirus is used as an expressionvector, polynucleotide sequences encoding collagen or collagen-likepolypeptides may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing the encoded polypeptidesin infected hosts; see, e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA81:3655-3659 (1984). Alternatively, the vaccinia 7.5 K promoter may beused; see, e.g., Mackett et al. (1982) Proc. Natl. Acad. Sci. USA79:7415-7419; Mackett et al. (1982) J. Virol. 49:857-864; and Panicaliet al. (1982) Proc. Natl. Acad. Sci. USA 79:4927-4931.

A preferred expression system in mammalian host cells is the SemlikiForest virus. Infection of mammalian host cells, for example, babyhamster kidney (BHK) cells and Chinese hamster ovary (CHO) cells canyield very high recombinant expression levels. Semliki Forest virus is apreferred expression system as the virus has a broad host range suchthat infection of mammalian cell lines will be possible. Morespecifically, Semliki Forest virus can be used in a wide range of hosts,as the system is not based on chromosomal integration, and thus providesan easier way of obtaining modifications of the recombinant animalcollagens in studies aiming at identifying structure functionrelationships and testing the effects of various hybrid molecules.Methods for constructing Semliki Forest virus vectors for expression ofexogenous proteins in mammalian host cells are described in, forexample, Olkkonen et al. (1994) Methods Cell Biol 43:43-53.

Non-human Transgenic animals may also be used to express thepolypeptides of the present invention. Such systems can be constructedby operably linking the polynucleotide of the invention to a promoter,along with other required or optional regulatory sequences capable ofeffecting expression in mammary glands. Likewise, required or optionalpost-translational enzymes may be produced simultaneously in the targetcells employing suitable expression systems. Methods of using non-humantransgenic animals to recombinantly produce proteins are known in theart; See, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,824,838; U.S.Pat. No. 5,487,992; and U.S. Pat. No. 5,614,396.

The references cited in the sections above which describe the productionof recombinant collagens are each incorporated by reference.

Composite collagen fiber sheets. As shown in FIG. 1, triple helicalcollagen molecules associate into fibrils which in animal skin assembleinto larger fibril bundles or collagen fibers. Prior methods of makingcollagen sheets used a mixture of ground animal skin or leather scrapsand dissolved or suspended collagen. Such collagen fiber-containingproducts are described by U.S. Pat. Nos. 2,934,446; 3,073,714; 3,122,599; and 3,136,682. Highberger, et al., U.S. Pat. No. 2,934,446describes a method using a meat grinder to produce a slurry of calfskinhide or corium which is formed into a sheet, tanned and for forminginterlocked collagen fiber masses by comminuting and dispersing animalskin in an acidic aqueous solution at 5° C. and then raising the pH andtemperature to precipitate collagen fibers to form a gel which is thendried. These sheets of collagen fiber masses make use of leather scrapsand form sheets resembling leather. Highberger does not show that theseleather sheets are suitable for commercial use. Tu, et al., U.S. Pat.No. 3,073,714 discloses producing a sheet from an calfskin slurrycontaining 25% solids which is tanned with a vegetable tanning solutionand treated with glycerin and oleic acid. These collagen fiber sheetsare described as reproducing the internal arrangement of collagen fibersin natural skins and hides. Tu does not show that the leather sheets arecompositionally or aesthetically suitable for use in a consumer product.Tu, et al., U.S. Pat. No. 3,122,599 describes a leather-like sheet madefrom ground animal skin or leather which contains collagen fibers andsoluble collagen as well as other components derived from the animalskin. Tu discloses treating this mixture with chromium, dehydrating itwith acetone, and treating with oleic acid to produce a leather-likeproduct containing collagen fiber masses. Tu does not show that thesheet is compositionally, physically or aesthetically suitable for usein a consumer product. Tu, et al., U.S. Pat. No. 3,136,682 describes aprocess of making a leather-like material that contains a mixture ofcollagen fibers and a binder of water-soluble proteinaceous materialderived from animal skin. It also describes the use of a chromiumtanning agent and treatment with oleic acid. Tu describes a sheet ofgood appearance and feel, but does not show that it is suitable forincorporation into a consumer product. These products incorporatecoarse, ground or digested collagen fibers.

Cultured leather products. These products generally comprise a pluralityof layers containing collagen produced by culturing cells in vitro aredescribed by Forgacs, et al., U.S. 2016/0097109 A1 and by Greene, U.S.Pat. No. 9,428,817 B2. These products are produced in vitro bycultivation of cell explants or cultured collagen-producing cells. Suchcells produce and process collagen into quaternary bundles of collagenfibrils and do not have the random, non-antistrophic structure of thecollagen fibrils of the invention. Forgacs describes engineered animalskins, which may be shaped, to produce a leather product. Greendescribes a variety of products, such as footwear, apparel and luggagethat may incorporate leather that is cultured in vitro. US 2013/0255003describes producing collagen for leather-like products by growing bovineskin cells in culture. Other types of host cells have been utilized toproduce collagen for medical implants or to produce gelatin. Forexample, United States Patent Application US 2004/0018592 describes away to produce gelatin by recombinantly expressing bovine collagen inhost cells, such as yeast.

Medical products. Networks of collagen have been produced in vitro asmaterials for biomedical applications. In those applications, monomersof the collagen triple helix are extracted from animal tissue, such asbovine dermis, either by acid treatment or treatment with proteindegrading enzymes such as pepsin, to solubilize collagen from thetissue. Once purified, these solubilized collagens (often mixtures ofmonomers, dimers and trimers of the collagen triple helix) can befibrillated into fibrils through a pH shift in aqueous buffers. Underthe right conditions, the collagen monomers self-assemble into fibrils,and depending on their source and how they were isolated, the fibrilscan physically crosslink to form a solid hydrogel. In addition,recombinant collagens and collagen-like proteins have been shown tofibrillate in vitro through similar adjustments in pH and saltconcentration. Examples of such products for medical applicationsinclude a biodegradable collagen matrix made from a collagen slurry thatself-assembles into macroscopic collagen fibers, U.S. Pat. No.9,539,363, and an organized array of collagen fibrils produced by use ofexternal guidance structures or internal templates and the applicationof tension, U.S. Pat. No. 9,518,106. Collagen products used in medicine,such as for tissue engineering or grafting, often aim to providecollagen in a form similar to that in a particular tissue beingengineered or repaired. While fibrillation of soluble collagens andcollagen-like proteins has been explored to produce collagen hydrogelsfor biomedical applications, this technology has not been successfullyapplied to the production of a material having the strength andaesthetic properties of natural leather.

Synthetic plastic-based leathers. Attempts to create synthetic leatherhave come up short in reproducing leather's unique set of functional andaesthetic properties. Examples of synthetic leather materials includeClarino, Naugahyde®, Corfam, and Alcantara, amongst others. They aremade of various chemical and polymer ingredients, including polyvinylchloride, polyurethane, nitrocellulose coated cotton cloth, polyester,or other natural cloth or fiber materials coated with a syntheticpolymer. These materials are assembled using a variety of techniques,often drawing from chemical and textile production approaches, includingnon-woven and advanced spinning processes. While many of these materialshave found use in footwear, upholstery, and apparel applications, theyhave fallen short for luxury application, as they cannot match thebreathability, performance, hand feel, or aesthetic properties that makeleather so unique and beloved. To date, no alternative commercialleather-like materials have been made from a uniform network of collagenor collagen-like proteins. Synthetic plastic materials lack the chemicalcomposition and structure of a collagen network that produces anacceptable leather aesthetic. Unlike, synthetics, the chemicalcomposition of amino acid side groups along the collagen polypeptidechain, along with its organization into a strong yet porous, fibrousarchitecture allow stabilization and functionalization of the fibrilnetwork through crosslinking processes to produce the desirablestrength, softness and aesthetic of leather.

While fibrillation of soluble collagens and collagen-like proteins hasbeen explored to bind together ground or comminuted leather scraps orfor the production of collagen hydrogels for biomedical applications,harnessing this phenomenon to produce a commercially acceptableleather-like material has not been achieved.

In view of the problems with prior art natural leathers, and composite,cultured, and synthetic, plastic-based leather products the inventorsdiligently pursued a way to provide a biofabricated leather havingsuperior strength and uniformity and non-anisotropic properties thatincorporated natural components found in leather.

Described herein are materials composed of collagen fibrils fibrillatedin vitro that have leather-like properties imparted by crosslinking,dehydration and lubrication. Compared to tanned and fatliquored animalhides, these biofabricated materials can have structural, compositionaland functional uniformity, for example, advantageous substantiallynon-anisotropic strength and other mechanical properties as well as atop grain like aesthetic on both their top and bottom surfaces.

SUMMARY OF THE INVENTION

The invention is directed to composite materials which incorporate abiofabricated material as described herein. The composites of theinvention include those where (i) one or more secondary components, suchas a particle, wire, fabric, or three dimensional object is incorporatedor embedded in a network of collagen fibrils, (ii) where a biofabricatedmaterial is coated or deposited, for example by filtration, on one sideof one or more secondary components such as a woven or nonwoven fabric,such as fabric, paper or regenerated cellulose, (iii) where abiofabricated component is coated or deposited on both sides of one ormore secondary materials having top and bottom sides or inner and outersides, or (iv) where a biofabricated material component and one or moresecondary components are adhered, attached or laminated to each other,for example, by direct lamination with or without an adhesive.

The composites of the invention contain a biofabricated materialcomponent. This component is composed of a network of crosslinked andlubricated collagen fibrils. It may be produced from collagen isolatedfrom an animal source or recombinant collagen. It can be produced fromcollagens that contain substantially no residues. Preferably it issubstantially free of large bundles of collagen fibers or othernon-hydroxylysine collagen components of leather, such as elastin. Thismaterial is composed of collagen which is also a major component ofnatural leather and is produced by a process of fibrillation of collagenmolecules into fibrils, crosslinking the fibrils and lubricating thecrosslinked fibrils. Unlike natural leathers, this biofabricatedmaterial exhibits non-anisotropic (not directionally dependent) physicalproperties, for example, a sheet of biofabricated material can havesubstantially the same elasticity or tensile strength when measured indifferent directions. Unlike natural leather, it has a uniform texturethat facilitates uniform uptake of dyes and coatings. Aesthetically, itproduces a uniform and consistent grain for ease of manufacturability.Composite materials incorporating this biofabricated material can havesubstantially identical grain, texture and other aesthetic properties onboth sides unlike natural leathers where the grain increases from oneside (e.g., distal surface) to the other (proximal inner layers).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing the composition of collagen in ahierarchical fashion. Reference character (1) shows each triple helicalcollagen monomer and how they are assembled with respect to neighboringcollagen monomers; (2) shows assembled collagen that makes up bandedcollagen fibrils; (3) shows the collagen fibrils at larger scale; (4)shows collagen fibrils aligned into fibers; and (5) shows bundles ofcollagen fibers.

FIG. 2A is a picture showing the composition of buffalo hide. The topgrain layer and the corium layer underneath are shown and the relativedegrees of higher order organization from collagen fibrils to collagenfiber bundles are indicated. The top grain layer is mostly composed offine collagen fibrils while the corium layer is mostly composed ofcoarser collagen fibers and fiber bundles.

FIGS. 2B and 2C compare the textures and grains of the outer and innersurfaces of leather depicting fine grain on one side and coarser coriumon the other.

FIG. 3A is a scanning electron micrograph of the fibrillated collagenhydrogel showing a network of fine collagen fibrils.

FIG. 3B is a scanning electron micrograph of bovine corium showingcoarser fiber bundles.

FIG. 4 is a transmission electron micrograph of a fibrillated collagennetwork or hydrogel showing fibril banding.

DETAILED DESCRIPTION OF THE INVENTION

“Biofabricated material” or “biofabricated leather” as used herein is amaterial produced from collagen or a collagen-like protein. It can beproduced from non-human collagens such as bovine, buffalo, ox, dear,sheep, goat, or pig collagen, which may be isolated from a naturalsource like animal hide, by in vitro culture of mammalian or animalcells, recombinantly produced or chemically synthesized. It is not aconventional material or leather which is produced from animal skins.Methods for producing this biofabricated material or biofabricatedleather are disclosed herein and usually involve fibrillating anisolated or purified solution or suspension of collagen molecules toproduce collagen fibrils, crosslinking the fibrils, dehydrating thefibrils and lubricating the fibrils.

In contrast to natural leathers which exhibit heterogeneous internalcollagen structures, a biofabricated material or biofabricated leathercan exhibit a substantially uniform internal structure characterized byunbundled and randomly-oriented collagen fibrils throughout its volume.

The resulting biofabricated material may be used in any way that naturalleather is used and may be grossly similar in appearance and feel toreal leather, while having compositional, functional or aestheticfeatures that differentiate it from ordinary leather. For example,unlike natural leather, a biofabricated leather need not containpotentially allergenic non-collagen proteins or components found in anatural leather, a biofabricated leather may exhibit a similarflexibility and strength in all directions (non-anisotropy) due tosubstantial non-alignment of its collagen fibrils, and aesthetically mayhave a smooth grain texture on both sides. A biofabricated leather canexhibit uniformity of properties including uniform thickness andconsistency, uniform distribution of lubricants, crosslinkers and dyes,uniform non-anisotropic strength, stretch, flexibility and resistance topiping (or the tendency for natural leather to separate or splitparallel to a plane of a sheet). By selecting the content of collagenand processing conditions, biofabricated leather can be “tuned” to aparticular thickness, consistency, flexibility, softness, and drapesurface texture or other functionality. Laminated, layered or compositeproducts may comprise a biofabricated leather.

A “composite” is a combination of a biofabricated material orbiofabricated leather component and a secondary material. The secondarycomponent may be incorporated into the biofabricated material; thebiofabricated material may be at least partially incorporated into asecondary material, or coated on, layered on, or laminated to asecondary material. Examples of composites include a biofabricatedmaterial encapsulating a secondary material, a secondary material coatedon one side with a biofabricated material, a secondary material coatedon both external sides with a biofabricated material, and one or morelayers of a secondary material laminated to one or more layers of abiofabricated material. This term encompasses all forms and combinationsof a biofabricated material and one or more secondary materials.

The term “collagen” refers to any one of the known collagen types,including collagen types I through XX, as well as to any othercollagens, whether natural, synthetic, semi-synthetic, or recombinant.It includes all of the collagens, modified collagens and collagen-likeproteins described herein. The term also encompasses procollagens andcollagen-like proteins or collagenous proteins comprising the motif(Gly-X—Y)n where n is an integer. It encompasses molecules of collagenand collagen-like proteins, trimers of collagen molecules, fibrils ofcollagen, and fibers of collagen fibrils. It also refers to chemically,enzymatically or recombinantly-modified collagens or collagen-likemolecules that can be fibrillated as well as fragments of collagen,collagen-like molecules and collagenous molecules capable of assemblinginto a nanofiber.

In some embodiments, amino acid residues, such as lysine and proline, ina collagen or collagen-like protein may lack hydroxylation or may have alesser or greater degree of hydroxylation than a corresponding naturalor unmodified collagen or collagen-like protein. In other embodiments,amino acid residues in a collagen or collagen-like protein may lackglycosylation or may have a lesser or greater degree of glycosylationthan a corresponding natural or unmodified collagen or collagen-likeprotein.

The collagen in a collagen composition may homogenously contain a singletype of collagen molecule, such as 100% bovine Type I collagen or 100%Type III bovine collagen, or may contain a mixture of different kinds ofcollagen molecules or collagen-like molecules, such as a mixture ofbovine Type I and Type III molecules. Such mixtures may include >0%, 10,20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or <100% of the individualcollagen or collagen-like protein components. This range includes allintermediate values. For example, a collagen composition may contain 30%Type I collagen and 70% Type III collagen, or may contain 33.3% of TypeI collagen, 33.3% of Type II collagen, and 33.3% of Type III collagen,where the percentage of collagen is based on the total mass of collagenin the composition or on the molecular percentages of collagenmolecules.

“Collagen fibrils” are nanofibers composed of tropocollagen (triplehelices of collagen molecules). Tropocollagens also includetropocollagen-like structures exhibiting triple helical structures. Thecollagen fibrils of the invention may have diameters ranging from 1 nmand 1 μm. For example, the collagen fibrils of the invention may have anaverage or individual fibril diameter ranging from 1, 2, 3, 4, 5, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, or 1,000 nm (1 μm). This range includes all intermediatevalues and subranges. In some of the embodiments of the inventioncollagen fibrils will form networks, for example, as depicted by FIGS. 3and 4. Collagen fibrils can associate into fibrils exhibiting a bandedpattern as shown in FIG. 1 and these fibrils can associate into largeraggregates of fibrils. In some embodiments the collagen or collagen-likefibrils will have diameters and orientations similar to those in the topgrain or surface layer of a bovine or other conventional leather. Inother embodiments, the collagen fibrils may have diameters comprisingthe top grain and those of a corium layer of a conventional leather.

A “collagen fiber” is composed of collagen fibrils that are tightlypacked and exhibit a high degree of alignment in the direction of thefiber as shown in FIG. 1. It can vary in diameter from more than 1 μm tomore than 10 μm, for example >1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 μmor more. Some embodiments of the network of collage fibrils of theinvention do not contain substantial content of collagen fibers havingdiameters greater than 5 μm. As shown in FIG. 2, the composition of thegrain surface of a leather can differ from its more internal portions,such as the corium which contains coarser fiber bundles.

“Fibrillation” refers to a process of producing collagen fibrils. It maybe performed by raising the pH or by adjusting the salt concentration ofa collagen solution or suspension. In forming the fibrillated collagen,the collagen may be incubated to form the fibrils for any appropriatelength of time, including between 1 min and 24 hrs and all intermediatevalues.

The fibrillated collagen described herein may generally be formed in anyappropriate shape and/or thickness, including flat sheets, curvedshapes/sheets, cylinders, threads, and complex shapes. These sheets andother forms may have virtually any linear dimensions including athickness, width or height greater of 10, 20, 30, 40, 50, 60, 70,80, 90mm; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 500, 1,000, 1,500,2,000 cm or more.

The fibrillated collagen in a biofabricated leather may lack any or anysubstantial amount of higher order structure. In a preferred embodiment,the collagen fibrils in a biofabricated leather will be unbundled andnot form the large collagen fibers found in animal skin and provide astrong and uniform non-anisotropic structure to the biofabricatedleather.

In other embodiments, some collagen fibrils can be bundled or alignedinto higher order structures. Collagen fibrils in a biofabricatedleather may exhibit an orientation index ranging from 0, >0, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, <1.0, or 1.0, wherein an orientationindex of 0 describes collagen fibrils that lack alignment with otherfibrils and an orientation index of 1.0 describes collagen fibrils thatare completely aligned. This range includes all intermediate values andsubranges. Those of skill in the art are familiar with the orientationindex which is also incorporated by reference to Sizeland, et al., J.Agric. Food Chem. 61: 887-892 (2013) or Basil-Jones, et al., J. Agric.Food Chem. 59: 9972-9979 (2011).

The methods disclosed herein make it possible to produce a biofabricatedleather comprising collagen fibrils differing in diameter from thoseproduced by an animal expressing the same type of collagen. Thecharacteristics of natural collagens, such as fibril diameter and degreeof crosslinking between fibrils are affected by genetic andenvironmental factors such as the species or breed of the animal and bythe condition of the animal, for example the amount of fat, type of feed(e.g. grain, grass), and level of exercise.

A biofabricated leather may be fibrillated and processed to containcollagen fibrils that resemble or mimic the properties of collagenfibrils produced by particular species or breeds of animals or byanimals raised under particular conditions.

Alternatively, fibrillation and processing conditions can be selected toprovide collagen fibrils distinct from those found in nature, such as bydecreasing or increasing the fibril diameter, degree of alignment, ordegree of crosslinking compared to fibrils in natural leather.

A crosslinked network of collagen, sometimes called a hydrogel, may beformed as the collagen is fibrillated, or it may form a network afterfibrillation; in some variations, the process of fibrillating thecollagen also forms gel-like network. Once formed, the fibrillatedcollagen network may be further stabilized by incorporating moleculeswith di-, tri-, or multifunctional reactive groups that includechromium, amines, carboxylic acids, sulfates, sulfites, sulfonates,aldehydes, hydrazides, sulfhydryls, diazarines, aryl-, azides,acrylates, epoxides, or phenols.

The fibrillated collagen network may also be polymerized with otheragents (e.g. polymers that are capable of polymerizing or other suitablefibers), which could be used to further stabilize the matrix and providethe desired end structure. Hydrogels based upon acrylamides, acrylicacids, and their salts may be prepared using inverse suspensionpolymerization. Hydrogels described herein may be prepared from polarmonomers. The hydrogels used may be natural polymer hydrogels, syntheticpolymer hydrogels, or a combination of the two. The hydrogels used maybe obtained using graft polymerization, crosslinking polymerization,networks formed of water soluble polymers, radiation crosslinking, andso on. A small amount of crosslinking agent may be added to the hydrogelcomposition to enhance polymerization.

Average or individual collagen fibril length may range from 100, 200,300, 400, 500, 600, 700, 800, 900, 1,000 (1 μm); 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 μm (1mm) throughout the entire thickness of a biofabricated leather. Theseranges include all intermediate values and subranges.

Fibrils may align with other fibrils over 50, 100, 200, 300, 400, 500 μmor more of their lengths or may exhibit little or no alignment. In otherembodiments, some collagen fibrils can be bundled or aligned into higherorder structures.

Collagen fibrils in a biofabricated leather may exhibit an orientationindex ranging from 0, >0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,<1.0, or 1.0, wherein an orientation index of 0 describes collagenfibrils that lack alignment with other fibrils and an orientation indexof 1.0 describes collagen fibrils that are completely aligned. Thisrange includes all intermediate values and subranges. Those of skill inthe art are familiar with the orientation index which is alsoincorporated by reference to Sizeland, et al., J. Agric. Food Chem. 61:887-892 (2013) or Basil-Jones, et al., J. Agric. Food Chem. 59:9972-9979 (2011).

Collagen fibril density of a biofabricated leather may range from about1 to 1,000 mg/cc, preferably from 5 to 500 mg/cc including allintermediate values, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1,000mg/cc.

The collagen fibrils in a biofabricated leather may exhibit a unimodal,bimodal, trimodal, or multimodal distribution, for example, abiofabricated leather may be composed of two different fibrilpreparations each having a different range of fibril diameters arrangedaround one of two different modes. Such mixtures may be selected toimpart additive, synergistic or a balance of physical properties on abiofabricated leather conferred by fibrils having different diameters.

Natural leather products may contain 150-300 mg/cc collagen based on theweight of the leather product. A biofabricated leather may contain asimilar content of collagen or collagen fibrils as conventional leatherbased on the weight of the biofabricated leather, such as a collagenconcentration of 100, 150, 200, 250, 300 or 350 mg/cc.

The fibrillated collagen, sometimes called a hydrogel, may have athickness selected based on its ultimate use. Thicker or moreconcentrated preparations of the fibrillated collagen generally producethicker biofabricated leathers. The final thickness of a biofabricatedleather may be only 10, 20, 30, 40, 50, 60, 70, 80 or 90% that of thefibril preparation prior to shrinkage caused by crosslinking,dehydration and lubrication.

“Crosslinking” refers to formation (or reformation) of chemical bondswithin between collagen molecules. A crosslinking reaction stabilizesthe collagen structure and in some cases forms a network betweencollagen molecules. Any suitable crosslinking agent known in the art canbe used including, without limitation, mineral salts such as those basedon chromium, formaldehyde, hexamethylene diisocyanate, glutaraldehyde,polyepoxy compounds, gamma irradiation, and ultraviolet irradiation withriboflavin. The crosslinking can be performed by any known method; see,e.g., Bailey et al., Radiat. Res. 22:606-621 (1964); Housley et al.,Biochem. Biophys. Res. Commun. 67:824-830 (1975); Siegel, Proc. Natl.Acad. Sci. U.S.A. 71:4826-4830 (1974); Mechanic et al., Biochem.Biophys. Res. Commun. 45:644-653 (1971); Mechanic et al., Biochem.Biophys. Res. Commun. 41:1597-1604 (1970); and Shoshan et al., Biochim.Biophys. Acta 154:261-263 (1968) each of which is incorporated byreference.

Crosslinkers include isocyantes, carbodiimide, poly(aldehyde),poly(azyridine), mineral salts, poly(epoxies), enzymes, thiamine,phenolics, novolac, resole as well as other compounds that havechemistries that react with amino acid side chains such as lysine,arginine, aspartic acid, glutamic acid, hydroxylproline, orhydroxylysine.

A collagen or collagen-like protein may be chemically modified topromote chemical and/or physical crosslinking between the collagenfibrils. Chemical crosslinking may be possible because reactive groupssuch as lysine, glutamic acid, and hydroxyl groups on the collagenmolecule project from collagen's rod-like fibril structure. Crosslinkingthat involve these groups prevent the collagen molecules from slidingpast each other under stress and thus increases the mechanical strengthof the collagen fibers. Examples of chemical crosslinking reactionsinclude but are not limited to reactions with the c-amino group oflysine, or reaction with carboxyl groups of the collagen molecule.Enzymes such as transglutaminase may also be used to generate crosslinksbetween glutamic acid and lysine to form a stable y-glutamyl-lysinecrosslink. Inducing crosslinking between functional groups ofneighboring collagen molecules is known in the art. Crosslinking isanother step that can be implemented here to adjust the physicalproperties obtained from the fibrillated collagen hydrogel-derivedmaterials.

Still fibrillating or fibrillated collagen may be crosslinked orlubricated. Collagen fibrils can be treated with compounds containingchromium or at least one aldehyde group, or vegetable tannins prior tonetwork formation, during network formation, or network gel formation.Crosslinking further stabilizes the fibrillated collagen leather. Forexample, collagen fibrils pre-treated with acrylic polymer followed bytreatment with a vegetable tannin, such as Acacia Mollissima, canexhibit increased hydrothermal stability. In other embodiments,glyceraldehyde may be used as a cross-linking agent to increase thethermal stability, proteolytic resistance, and mechanicalcharacteristics, such as Young's modulus and tensile stress, of thefibrillated collagen.

A biofabricated material containing a network of collagen fibrils maycontain 0, >0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20% or more of acrosslinking agent including tanning agents used for conventionalleather. The crosslinking agents may be covalently bound to the collagenfibrils or other components of a biofabricated material ornon-covalently associated with them. Preferably, a biofabricated leatherwill contain no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% of acrosslinking agent.

“Lubricating” describes a process of applying a lubricant, such as a fator other hydrophobic compound or any material that modulates or controlsfibril-fibril bonding during dehydration to leather or to biofabricatedproducts comprising collagen. A desirable feature of the leatheraesthetic is the stiffness or hand of the material. In order to achievethis property, water-mediated hydrogen bonding between fibrils and/orfibers is limited in leather through the use of lubricants. Examples oflubricants include fats, biological, mineral or synthetic oils, cod oil,sulfonated oil, polymers, organofunctional siloxanes, and otherhydrophobic compounds or agents used for fatliquoring conventionalleather as well as mixtures thereof. While lubricating is in some waysanalogous to fatliquoring a natural leather, a biofabricated product canbe more uniformly treated with a lubricant due to its method ofmanufacture, more homogenous composition and less complex composition.

Other lubricants include surfactants, anionic surfactants, cationicsurfactants, cationic polymeric surfactants, anionic polymericsurfactants, amphiphilic polymers, fatty acids, modified fatty acids,nonionic hydrophilic polymers, nonionic hydrophobic polymers, polyacrylic acids, poly methacrylic, acrylics, natural rubbers, syntheticrubbers, resins, amphiphilic anionic polymer and copolymers, amphiphiliccationic polymer and copolymers and mixtures thereof as well asemulsions or suspensions of these in water, alcohol, ketones, and othersolvents.

Lubricants may be added to a biofabricated material containing collagenfibrils. Lubricants may be incorporated in any amount that facilitatesfibril movement or that confers leather-like properties such asflexibility, decrease in brittleness, durability, or water resistance. Alubricant content can range from about 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60% by weight ofthe biofabricated leather.

“Dehydrating” or “dewatering” describes a process of removing water froma mixture containing collagen fibrils and water, such as an aqueoussolution, suspension, gel, or hydrogel containing fibrillated collagen.Water may be removed by filtration, evaporation, freeze-drying, solventexchange, vacuum-drying, convection-drying, heating, irradiating ormicrowaving, or by other known methods for removing water. In addition,chemical crosslinking of collagen is known to remove bound water fromcollagen by consuming hydrophilic amino acid residues such as lysine,arginine, and hydroxylysine among others. The inventors have found thatacetone quickly dehydrates collagen fibrils and may also remove waterbound to hydrated collagen molecules. Water content of a biofabricatedmaterial or leather after dehydration is preferably no more than 60% byweight, for example, no more than 5, 10, 15, 20, 30, 35, 40, 50 or 60%by weight of the biofabricated leather. This range includes allintermediate values. Water content is measured by equilibration at 65%relative humidity at 25° C. and 1 atm.

“Grain texture” describes a leather-like texture which is aestheticallyor texturally the similar to the texture of a full grain leather, topgrain leather, corrected grain leather (where an artificial grain hasbeen applied), or coarser split grain leather texture. Advantageously,the biofabricated material of the invention can be tuned to provide afine grain, resembling the surface grain of a leather such as thatdepicted by FIGS. 2A, 2B and 2C.

A “biofabricated leather product” includes products comprising at leastone component of a biofabricated leather such as foot ware, garments,gloves, furniture or vehicle upholstery and other leather goods andproducts. It includes but is not limited to clothing, such as overcoats,coats, jackets, shirts, trousers, pants, shorts, swimwear,undergarments, uniforms, emblems or letters, costumes, ties, skirts,dresses, blouses, leggings, gloves, mittens, foot ware, shoes, shoecomponents such as sole, quarter, tongue, cuff, welt, and counter, dressshoes, athletic shoes, running shoes, casual shoes, athletic, running orcasual shoe components such as toe cap, toe box, outsole, midsole,upper, laces, eyelets, collar, lining, Achilles notch, heel, andcounter, fashion or women's shoes and their shoe components such asupper, outer sole, toe spring, toe box, decoration, vamp, lining, sock,insole, platform, counter, and heel or high heel, boots, sandals,buttons, sandals, hats, masks, headgear, headbands, head wraps, andbelts; jewelry such as bracelets, watch bands, and necklaces; gloves,umbrellas, walking sticks, wallets, mobile phone or wearable computercoverings, purses, backpacks, suitcases, handbags, folios, folders,boxes, and other personal objects; athletic, sports, hunting orrecreational gear such as harnesses, bridles, reins, bits, leashes,mitts, tennis rackets, golf clubs, polo, hockey, or lacrosse gear,chessboards and game boards, medicine balls, kick balls, baseballs, andother kinds of balls, and toys; book bindings, book covers, pictureframes or artwork; furniture and home, office or other interior orexterior furnishings including chairs, sofas, doors, seats, ottomans,room dividers, coasters, mouse pads, desk blotters, or other pads,tables, beds, floor, wall or ceiling coverings, flooring; automobile,boat, aircraft and other vehicular products including seats, headrests,upholstery, paneling, steering wheel, joystick or control coverings andother wraps or coverings.

Many uses of leather products require a durable product that doesn't ripor tear, even when the leather has been stitched together. Typicalproducts that include stitched leather and require durable leatherinclude automobile steering wheel covers, automobile seats, furniture,sporting goods, sport shoes, sneakers, watch straps and the like. Thereis a need to increase the durability of biofabricated leather to improveperformance in these products. A biofabricated leather according to theinvention can be used to make any of these products.

Physical Properties of a biofabricated network of collagen fibrils or abiofabricated leather may be selected or tuned by selecting the type ofcollagen, the amount of concentration of collagen fibrillated, thedegree of fibrillation, crosslinking, dehydration and lubrication. Manyadvantageous properties are associated with the network structure of thecollagen fibrils which can provide strong, flexible and substantiallyuniform properties to the resulting biofabricated material or leather.Preferable physical properties of the biofabricated leather according tothe invention include a tensile strength ranging from 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15 or more MPa, a flexibility determined byelongation at break ranging from 1, 5, 10, 15, 20, 25, 30% or more,softness as determined by ISO 17235 of 4, 5, 6, 7, 8 mm or more, athickness ranging from 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,1.2, 1.3. 1,4, 1,5, 1.6, 1.7, 1.8, 1.9, 2.0 mm or more, and a collagendensity (collagen fibril density) of 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 mg/cc or more,preferably 100-500 mg/cc. The above ranges include all subranges andintermediate values.

11371 Thickness. Depending on its ultimate application a biofabricatedmaterial or leather may have any thickness. Its thickness preferablyranges from about 0.05 mm to 20 mm as well as any intermediate valuewithin this range, such as 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 mm ormore. The thickness of a biofabricated leather can be controlled byadjusting collagen content.

Elastic modulus. The elastic modulus (also known as Young's modulus) isa number that measures an object or substance's resistance to beingdeformed elastically (i.e., non-permanently) when a force is applied toit. The elastic modulus of an object is defined as the slope of itsstress-strain curve in the elastic deformation region. A stiffermaterial will have a higher elastic modulus. The elastic modulus can bemeasured using a texture analyzer.

A biofabricated leather can have an elastic modulus of at least 100 kPa.It can range from 100 kPa to 1,000 MPa as well as any intermediate valuein this range, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800,900, or 1,000 MPa. A biofabricated leather may be able to elongate up to300% from its relaxed state length, for example, by >0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or300% of its relaxed state length.

Tensile strength (also known as ultimate tensile strength) is thecapacity of a material or structure to withstand loads tending toelongate, as opposed to compressive strength, which withstands loadstending to reduce size. Tensile strength resists tension or being pulledapart, whereas compressive strength resists compression or being pushedtogether.

A sample of a biofabricated material may be tested for tensile strengthusing an Instron machine. Clamps are attached to the ends of the sampleand the sample is pulled in opposite directions until failure. Goodstrength is demonstrated when the sample has a tensile strength of atleast 1 MPa. A biofabricated leather can have a tensile strength of atleast 1 kPa. It can range from 1 kPa to 100 MPa as well as anyintermediate value in this range, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,50, 100, 200, 300, 400, 500 kPa; 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80,90, or 100 MPa.

Tear strength (also known as tear resistance) is a measure of how well amaterial can withstand the effects of tearing. More specifically howeverit is how well a material (normally rubber) resists the growth of anycuts when under tension, it is usually measured in kN/m. Tear resistancecan be measured by the ASTM D 412 method (the same used to measuretensile strength, modulus and elongation). ASTM D 624 can be used tomeasure the resistance to the formation of a tear (tear initiation) andthe resistance to the expansion of a tear (tear propagation). Regardlessof which of these two is being measured, the sample is held between twoholders and a uniform pulling force applied until the aforementioneddeformation occurs. Tear resistance is then calculated by dividing theforce applied by the thickness of the material. A biofabricated leathermay exhibit tear resistance of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 100, 150 or 200% more than that of aconventional top grain or other leather of the same thickness comprisingthe same type of collagen, e.g., bovine Type I or Type III collagen,processed using the same crosslinker(s) or lubricants. A biofabricatedmaterial may have a tear strength ranging from about 1 to 500 N, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425,450, 475, or 500 as well as any intermediate tear strength within thisrange.

Softness. ISO 17235:2015 specifies a non-destructive method fordetermining the softness of leather. It is applicable to all non-rigidleathers, e.g. shoe upper leather, upholstery leather, leather goodsleather, and apparel leather. A biofabricated leather may have asoftness as determined by ISO 17235 of 2, 3, 4, 5, 6, 7, 8, 10, 11, 12mm or more.

Grain. The top grain surface of leather is often regarded as the mostdesirable due to its soft texture and smooth surface. The top grain is ahighly porous network of collagen fibrils. The strength and tearresistance of the grain is often a limitation for practical applicationsof the top grain alone and conventional leather products are oftenbacked with corium having a much coarser grain. FIGS. 2A, 2B and 2Ccompare top grain and corium leather surfaces. A biofabricated materialas disclosed herein which can be produced with strong and uniformphysical properties or increased thickness can be used to provide topgrain like products without the requirement for corium backing.

Content of other components. In some embodiments, the collagen is freeof other leather components such as elastin or non-structural animalproteins. However, in some embodiments the content of actin, keratin,elastin, fibrin, albumin, globulin, mucin, mucinoids, noncollagenstructural proteins, and/or noncollagen nonstructural proteins in abiofabricated leather may range from 0, 1, 2, 3,4, 5, 6, 7, 8, 9 to 10%by weight of the biofabricated leather. In other embodiments, a contentof actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids,noncollagen structural proteins, and/or noncollagen nonstructuralproteins may be incorporated into a biofabricated leather in amountsranging from >0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20% or more by weight of a biofabricated leather. Suchcomponents may be introduced during or after fibrillation,cross-linking, dehydration or lubrication.

A “leather dye” refers to dyes which can be used to color leather orbiofabricated leather. These include acidic dyes, direct dyes, lakes,sulfur dyes, basic dyes and reactive dyes. Dyes and pigments can also beincorporated into a precursor of a biofabricated leather, such as into asuspension or network gel comprising collagen fibrils during productionof the biofabricated leather.

“Fillers”. In some embodiments a biofabricated leather may comprisefillers, other than components of leather, such as microspheres. One wayto control the organization of the dehydrated fibril network is toinclude filling materials that keep the fibrils spaced apart duringdehydration. These filler materials include nanoparticles,microparticles, or various polymers such as syntans commonly used in thetanning industry. These filling materials could be part of the finaldehydrated leather material, or the filling materials could besacrificial, that is they are degraded or dissolved away leaving openspace for a more porous fibril network. The shape and dimension of thesefillers may also be used to control the orientation of the dehydratedfibril network.

In some embodiments a filler or secondary component may comprisepolymeric microsphere(s), bead(s), fiber(s), wire(s), or organicsalt(s). Other materials may also be embedded or otherwise incorporatedinto a biofabricated leather or into a network of collagen fibrilsaccording to the invention. These include, but are not limited to onefibers, including both woven and nonwoven fibers as well as cotton,wool, cashmere, angora, linen, bamboo, bast, hemp, soya, seacell, fibersproduced from milk or milk proteins, silk, spider silk, other peptidesor polypeptides including recombinantly produced peptides orpolypeptides, chitosan, mycelium, cellulose including bacterialcellulose, wood including wood fibers, rayon, lyocell, vicose,antimicrobial yarn (A.M.Y.), Sorbtek, nylon, polyester, elastomers suchas lycra®, spandex or elastane and other polyester-polyurethanecopolymers, aramids, carbon including carbon fibers and fullerenes,glass including glass fibers and nonwovens, silicon andsilicon-containing compounds, minerals, including mineral particles andmineral fibers, and metals or metal alloys, including those comprisingiron, steel, lead, gold, silver, platinum, copper, zinc and titanium,which may be in the form of particles, fibers, wires or other formssuitable for incorporating into biofabricated leather. Such fillers mayinclude an electrically conductive material, magnetic material,fluorescent material, bioluminescent material, phosphorescent materialor other photoluminescent material, or combinations thereof. Mixtures orblends of these components may also be embedded or incorporated into abiofabricated leather, for example, to modify the chemical and physicalproperties disclosed herein.

Method of Making the Biofabricated Material Component of a Composite.

A method of forming biofabricated material component from collagen foruse in a composite material includes the steps of fibrillating,crosslinking, dehydrating/dewatering and lubricating in any order. Forexample, a collagen solution may be fibrillated, the fibrils may becrosslinked with an agent such as glutaraldehyde, then coated with alubricant such as a sulfited oil, and then dehydrated through filtrationto form a fibrillated collagen leather. However, the method of making isnot limited to this particular order of steps.

Alternatively, following fibril crosslinking, the fibrils can bedehydrated through a solvent exchange with acetone, followed by fatliquoring with a sulfited oil before evaporating away the solvent toform a fibrillated collagen leather. In addition, the incorporation ofchemical or physical crosslinks between fibrils (to impart materialstrength) can be accomplished at any point during the process. Forexample, a solid fibrillated collagen, sometimes called a hydrogel, canbe formed, then this fibril network can be dehydrated through a solventexchange with acetone, followed by fat liquoring with a sulfited oil.Further, the collagen fibrils can be crosslinked into a network throughthe incorporation of other polymers such as those typically used inresin formulations.

Materials such as lubricants, humectants, dyes and other treating agentscan be uniformly distributed through a biofabricated leather productduring the biofabrication process This is an advantage compared toconventional leather tanning and fat liquoring which due to itsstructural heterogeneity often makes uniform treatment impossible.Further, as chemical agents can be incorporated before networkformation, smaller amounts of treatment chemicals would be necessary asthere is reduced chemical loss by not having to penetrate a collagennetwork from a float containing the treatment chemicals. Unlike hightemperatures often used to treat natural leather, a biofabricated can beheated at ambient temperature or at a temperature no greater than 37° C.during processing before evaporating away the solvent to form afibrillated collagen leather. Alternatively, collagen fibrils can becrosslinked and lubricated in suspension before forming a networkbetween fibrils during dehydration or through the addition of a bindingagent to the suspension or to the dehydrated material.

A method of forming a biofabricated leather material may includeinducing fibrillation of collagen in a solution; crosslinking (e.g.,tanning) and dehydrating the fibrillated collagen, which may appear inthe form of a hydrogel, to obtain a fibrillated collagen sheet or otherproduct, and incorporating at least one humectant or lubricant, such asa fat or oil into the fibrillated collagen sheet or product to obtain aflexible biofabricated leather.

A method of biofabricating a leather from fibrils may include inducingfibrillation of collagen or collagen-like proteins in a solution toobtain a fibrillated collagen hydrogel; crosslinking the fibrillatedcollagen hydrogel to obtain a fibrillated collagen hydrogel leather; andincorporating at least one lubricating oil into the fibrillated collagenhydrogel leather.

In the processes described herein for producing a biofabricated leather,the order of the steps for forming biofabricated leather may be variedor two or more steps may be performed simultaneously. For example,fibrillating and crosslinking may be performed together or by additionof one or more agents, or crosslinker and lubricant may be incorporatedin the solution prior to fibrillating the collagen, etc.

The collagen or collagen-like proteins may be obtained throughextraction of collagen from an animal source, such as, but not limitedto bovine hide or tendon collagen extraction. Alternatively, thecollagen or collagen-like proteins may be obtained from a non-animalsource, for example through recombinant DNA techniques, cell culturetechniques, or chemical peptide synthesis.

Any of these methods may include polymerizing the collagen orcollagen-like proteins into dimers, trimers, and higher order oligomersprior to fibrillation, and/or chemically modifying the collagen orcollagen-like proteins to promote crosslinking between the collagen orcollagen-like proteins.

Any of these methods may include functionalizing the collagen orcollagen-like proteins with one or a combination of chromium, amine,carboxylic acid, sulfate, sulfite, sulfonate, aldehyde, hydrazide,sulfhydryl, diazirine, aryl, azide, acrylate, epoxide, or phenol group.

Inducing fibrillation may include adding a salt or a combination ofsalts, for example, the salt or combination of salts may include:Na₃PO₄, K₃PO₄, KCl, and NaCl, the salt concentration of each salt may bebetween 10 mM to 5M, etc.

In general, inducing fibrillation may comprise adjusting the pH with anacid or a base, adding a nucleating agent, such as a branched collagenmicrogel, wherein the nucleating agent has a concentration between 1 mMto 100 mM.

The fibrillated collagen may be stabilized with a chromium compound, analdehyde compound, or vegetable tannins, or any other crosslinkingagent. For example, the fibrillated collagen may be stabilized with achromium compound, an aldehyde compound, or vegetable tannins, whereinthe chromium, aldehyde, or vegetable tannin compounds having aconcentration of between 1 mM to 100 mM.

Any of these methods may include adjusting the water content of thefibrillated collagen to 5, 10, 20, 25, 30, 40, 50 or 60% or less byweight to obtain the fibrillated collagen hydrogel leather. For example,the fibrillated collagen material may be dehydrated. Any of thesemethods may also include dyeing and/or applying a surface finish to thefibrillated collagen leather.

The selection of collagen starting materials for biofabricating theengineered leather materials described herein can be controlled, theresulting product may differential formed with physical and aestheticproperties for distinct end uses, such as with features useful infootwear and different features useful in apparel. In general, thebiofabricated fibrillated collagen hydrogel-derived leathers describedherein are formed from solutions of collagen that are induced toself-assemble into collagen fibrils.

The collagen fibrils, unlike endogenous collagen fibrils, are notassembled into any high-order structures (e.g., bundles of fibers), butremain somewhat disordered, more particularly unbundled fibrils. Whenassembled in vivo, collagen fibrils are typically aligned laterally toform bundles having a higher order of structure and make up tough,micron-sized collagen fibers found, e.g., in skin. A characteristicfeature of native collagen fibrils is their banded structure. Thediameter of the native fibril changes slightly along the length, with ahighly reproducible D-band repeat of approximately 67 nm. In some of themethods described herein, collagen fibrils may be unbanded and unbundledor may be banded and unbundled or may have a D-band of different spacingranging from 1 to 100 nm and all intermediate values in this range). Thecollagen fibrils may be randomly oriented (e.g., un-oriented or notoriented in any particular direction or axis).

The starting material used to form the biofabricated leather material asdescribed herein may include any appropriate non-human collagen sourceor modified or engineered collagens that can be fibrillated.

Various forms of collagen are found throughout the animal kingdom. Thecollagen used herein may be obtained from animal sources, including bothvertebrates and invertebrates, or from synthetic sources. Collagen mayalso be sourced from byproducts of existing animal processing. Collagenobtained from animal sources may be isolated using standard laboratorytechniques known in the art, for example, Silva et. Al., Marine OriginCollagens and its Potential Applications, Mar. Drugs, 2014 December,12(12); 5881-5901).

One major benefit of the biofabricated leather materials and methods forforming them described herein is that collagen may be obtained fromsources that do not require killing of an animal.

The collagen described herein also may be obtained by cell culturetechniques including from cells grown in a bioreactor.

Collagen may also be obtained via recombinant DNA techniques. Constructsencoding non-human collagen may be introduced into host organisms toproduce non-human collagen. For instance, collagen may also be producedwith yeast, such as Hansenula polymorpha, Saccharomyces cerevisiae,Pichia pastoris and the like as the host. Further, in recent years,bacterial genomes have been identified that provide the signature(Gly-Xaa-Yaa)n repeating amino acid sequence that is characteristic oftriple helix collagen. For example, gram positive bacteriumStreptococcus pyogenes contains two collagen-like proteins, Scl1 andScl2 that now have well characterized structure and functionalproperties. Thus, it would be possible to obtain constructs inrecombinant E. coli systems with various sequence modifications ofeither Scl1 or Scl2 for establishing large scale production methods.Collagen may also be obtained through standard peptide synthesistechniques. Collagen obtained from any of the techniques mentioned maybe further polymerized. Collagen dimers and trimers are formed fromself-association of collagen monomers in solution.

As an initial step in the formation of the collagen materials describedherein, the starting collagen material may be placed in solution andfibrillated. Collagen fibrillation may be induced through theintroduction of salts to the collagen solution. The addition of a saltor a combination of salts such as sodium phosphate, potassium phosphate,potassium chloride, and sodium chloride to the collagen solution maychange the ionic strength of the collagen solution. Collagenfibrillation may occur as a result of increasing electrostaticinteractions, through greater hydrogen bonding, Van der Waalsinteractions, and covalent bonding. Suitable salt concentrations mayrange, for example, from approximately 10 mM, 50 mM, 100 mM, 500 mM, 1M,2M, 3M, 4M to 5M as well as any intermediate value within this range.

Collagen fibrillation may also be induced or enhanced with a nucleatingagent other than salts. Nucleating agents provide a surface on whichcollagen monomers can come into close contact with each other toinitiate fibrillation or can act as a branch point in which multiplefibrils are connected through the nucleating agent. Examples of suitablenucleating agents include but are not limited to: microgels containingcollagen, collagen micro or nanoparticles, metallic particles ornaturally or synthetically derived fibers. Suitable nucleating agentconcentrations may range from approximately 1 mM to 100 mM.

A collagen network may also be highly sensitive to pH. During thefibrillation step, the pH may be adjusted to control fibril dimensionssuch as diameter and length. The overall dimensions and organization ofthe collagen fibrils will affect the toughness, stretchability, andbreathability of the resulting fibrillated collagen derived materials.This may be of use for fabricating fibrillated collagen derived leatherfor various uses that may require different toughness, flexibility, andbreathability. Adjustment of pH, with or without a change in saltconcentration may be used for fibrillation.

One way to control the organization of the dehydrated fibril network isto include filling materials that keep the fibrils spaced apart duringdrying. These filler materials could include nanoparticles,microparticles, or various polymers such as syntans commonly used in thetanning industry. These filling materials could be part of the finaldehydrated leather material, or the filling materials could besacrificial, that is they are degraded or dissolved away leaving openspace for a more porous fibril network.

The collagen or collagen-like proteins may be chemically modified topromote chemical and physical crosslinking between the collagen fibrils.Chemical crosslinking may be possible because reactive groups such aslysine, glutamic acid, and hydroxyl groups on the collagen moleculeproject from collagen's rod-like fibril structure. Crosslinking thatinvolve these groups prevent the collagen molecules from sliding pasteach other under stress and thus increases the mechanical strength ofthe collagen fibers. Examples of chemical crosslinking reactions includebut are not limited to reactions with the ε-amino group of lysine, orreaction with carboxyl groups of the collagen molecule. Enzymes such astransglutaminase may also be used to generate crosslinks betweenglutamic acid and lysine to form a stable γ-glutamyl-lysine crosslink.Inducing crosslinking between functional groups of neighboring collagenmolecules is known in the art. Crosslinking is another step that can beimplemented here to adjust the physical properties obtained from thefibrillated collagen hydrogel-derived materials.

Once formed, the fibrillated collagen network may be further stabilizedby incorporating molecules with di-, tri-, or multifunctional reactivegroups that include chromium, amines, carboxylic acids, sulfates,sulfites, sulfonates, aldehydes, hydrazides, sulfhydryls, diazarines,aryl-, azides, acrylates, epoxides, or phenols.

The fibrillated collagen network may also be polymerized with otheragents (e.g. polymers that are capable of polymerizing or other suitablefibers) that form a hydrogel or have fibrous qualities, which could beused to further stabilize the matrix and provide the desired endstructure. Hydrogels based upon acrylamides, acrylic acids, and theirsalts may be prepared using inverse suspension polymerization. Hydrogelsdescribed herein may be prepared from polar monomers. The hydrogels usedmay be natural polymer hydrogels, synthetic polymer hydrogels, or acombination of the two. The hydrogels used may be obtained using graftpolymerization, crosslinking polymerization, networks formed of watersoluble polymers, radiation crosslinking, and so on. A small amount ofcrosslinking agent may be added to the hydrogel composition to enhancepolymerization.

Any appropriate thickness of the fibrillated collagen hydrogel may bemade as described herein. Because the final thickness will be much less(e.g., between 10-90% thinner) than the hydrogel thickness, the initialhydrogel thickness may depend on the thickness of the final productdesired, presuming the changes to the thickness (or overall volume)including shrinkage during crosslinking, dehydration and/or adding oneor more oils or other lubricants as described herein.

A hydrogel thickness may be between 0.1 mm and 50 cm or any intermediatevalue within this range. In forming the fibrillated hydrogel, thehydrogel may be incubated to form the thickness for any appropriatelength of time, including between 1 min and 24 hrs.

The fibrillated collagen hydrogels described herein may generally beformed in any appropriate shape and/or thickness, including flat sheets,curved shapes/sheets, cylinders, threads, and complex shapes. Further,virtually any linear size of these shapes. For example, any of thesehydrogels may be formed into a sheet having a thickness as described anda length of greater than 10 mm (e.g., greater than, in cm, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, etc.) and widththat is greater than 10 mm, such as greater than, in cm, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, etc.

Once the collagen fibrils, often characterized as a hydrogel, haveformed or during formation, they may be crosslinked. For example, thefibrillated collagen hydrogel be treated with compounds containingchromium or at least one aldehyde group, or vegetable tannins prior togel formation, during gel formation, or after gel formation, to furtherstabilize the fibrillated collagen hydrogel. For example, collagenfibrils may be pre-treated with acrylic polymer followed by treatmentwith a vegetable tannin (e.g., Acacia Mollissima) may exhibit increasedhydrothermal stability. In other examples, glyceraldehyde may be used asa cross-linking agent that may increase the thermal stability,proteolytic resistance, and mechanical characteristics (e.g. Young'smodulus, tensile stress) of the fibrillated collagen hydrogel.

Depending on the temperature and volume of starting material, thefibrillation and hydrogel formation may occur somewhat quickly afterinduction and be largely complete after an hour and a half, as shown bythe absorbance values leveling off after 70 minute's time passing. Anincrease in storage modulus (or viscoelastic qualities of the material)of the fibrillated collagen hydrogel after induction from around 1 Pa(for the solution of collagen) to approximately 400 Pa for thefibrillated collagen hydrogel may occur.

As mentioned above and illustrated in FIGS. 1 and 2, animal skintypically includes fibrils that are ordered into higher-orderstructures, including the presence of banding (having regular lacunarregions) and formation of multiple fibrils into aligned fibers which maythen bundled into collagen bundles. In contrast, the collagen hydrogelsand therefore the biofabricated leathers described herein may have aprimary disorganized collagen fibril structure throughout the entirethickness (in some cases entire volume) of the material. Specifically,the collagen structure of the biofabricated leathers formed fromcollagen hydrogels may be primarily unbundled and un-oriented along anyparticular axis. In some variations the collagen fibrils may be unbanded(e.g., greater than 10% unbanded, greater than 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, etc. unbanded throughout the volume). Furthermore, theorientation of the collagen fibrils within the volume (or throughout thevolume) may be un-oriented, or randomly oriented, and this lack oforientation may be the same throughout the volume, rather than changingthrough the volume thickness as in native leather, which may have changefrom bundles of collagen fibrils that are vertically oriented to bundlesthat are horizontally oriented in the thickness. Any of the propertieswhich are the same at any level thickness of the hydrogel and thereforeresulting leather material may be referred to herein as “uniformly” thesame throughout the thickness.

In addition, any of the biofabricated leathers described herein may havea uniform distribution of fibrils throughout the thickness of the geland therefore resulting leather material. This is in contrast withnative leathers, such as the material shown in FIG. 2, showing anincrease in the number of fiber bundles through the thickness of thematerial.

The lack of higher-level organization of the fibrillated collagenhydrogels and leather material formed from them is apparent in FIGS. 3Aand 3B. FIG. 3A shows a scanning electron micrograph of a fibrillatedcollagen hydrogel formed as described herein. Similarly, FIG. 4 shows atransmission electron micrograph through a fibrillated collagenhydrogel. The transmission electron micrograph and the scanning electronmicrograph both show the fibrillated collagen hydrogel as being adisordered tangle of collagen fibrils. As previously mentioned, thedensity and to some extent, the pattern of collagen fibril formation maybe controlled by adjusting the pH of the collagen solution duringfibrillation induction along with the concentration of fibrils duringdehydration. FIG. 3 also shows a scanning electron micrograph of bovinecorium. In comparison with a natural bovine corium shown in FIG. 3B, thefibrillated collagen network is much more random and lacks the apparentstriations. Although the overall size of the fibrils may be similar, thearrangement of these fibrils is quite different. Such ultrastructuraldifferences between the collagen fibrils within the fibrillated collagenhydrogel and natural tissue such as bovine corium (and resulting leathermade therefrom) may not be an issue in the final biofabricated leatherproduct may be as soft or softer, and more pliable than natural leather,and may have a similar appearance. In order to make the finalbiofabricated leather product more durable, the fibrillated collagen mayinclude a secondary material (collagen being the primary material).Suitable secondary materials include, but are not limited to, woven orknitted fabrics, nonwovens including natural felts such as wool feltsand the like, synthetic felts such as polyester-polyurethane copolymerssuch as elastane or LYCRA® felts, polyparaphenylene terephthalamidepolymers such as KEVLAR® felts, nylon polymers such as nylon 6, nylon6,6 and the like felts, and polyester polymers such as polyethyleneterephthalatepolyethylene and the like felts, staple fibers such ascarbon fibers felts, silk fibers and the like, cellulosic microfibersand combinations thereof. In one embodiment of the present invention,the secondary material is surrounded by the fibrillated collagenmaterial to create a composite. One method of surrounding a secondarymaterial with fibrillated collagen is to pour a collagen solution overone side of the secondary material, then the secondary material may beflipped and collagen solution poured onto the opposite side of thesecondary material. This may be described as a sandwich type structure.

In another embodiment of the present invention, the collagen may beconverted into a biofabricated leather and the secondary material may belaminated to one side of the leather using adhesives and the like.Suitable adhesives may include but are not limited to hot meltadhesives, emulsion polymer adhesives and the like. The biofabricatedleather may be coated with adhesive by known techniques such as slot diecasting, kiss coating and the like and the secondary material may beapplied to the leather and passed through rollers under heat to laminatethe materials.

In another embodiment, the secondary material may be dispersedthroughout the collagen material to create the composite structure. Thedensity of the secondary material may range from 1 μg/mL to 500 mg/mL.The ratio of fibrillated collagen to secondary material may range from1:100 to 100:1. The ratio of dried collagen to secondary material in thebiofabricated leather product may range from 1:100 to 100:1.

The secondary material may also be a photoluminescent material such as aphotoluminescent fabric, nonwoven, felt, carbon fiber or 3 dimensionalobject. As described above, the collagen solution may be poured over oneside of the secondary material, the secondary material may be flippedover and collagen solution may be poured over the other side of thesecondary material.

The fibrillated collagen, sometimes called a hydrogel, may then bedehydrated to rid the fibrillated collagen hydrogel of the majority ofits water content. Removing the water from the fibrillated collagenhydrogel may change its physical quality from a hydrated gel to apliable sheet. The material may be treated to prevent breakage/tearing.For example, care may be taken not to remove too much water from thefibrillated collagen. In some examples, it may be desirable to dehydratethe fibrillated collagen to have a water content of less than 5, 10, 15,20, 25, 30, 40, 50 or 60%. Water content is determined by equilibrationat 25° C. at 1 atm pressure at a relative humidity of 65%.

Dehydration may involve air drying, vacuum and pressure filtration,solvent exchange or the like. For example, fibrillated collagen hydrogelmay also undergo dehydration through replacement of its water contentwith organic solvents. Suitable organic solvents may include, but arenot limited to acetone, ethanol, diethyl ether, and so forth.Subsequently, the organic solvents may be evaporated (e.g. air drying,vacuum drying, etc.). It is also possible to perform successive steps ofdehydration using one or more than one organic solvent to fine tune thelevel of dehydration in the final product.

After or during dehydration, the fibrillated collagen material may betreated with lubricants and/or oils to impart greater flexibility andsuppleness to the fibrillated collagen material. Using a combination ofoil and solvent may allow the oil to better penetrate the fibrillatedcollagen network compared to using oil by itself. Oil by itself willonly likely penetrate the exposed surfaces but may not readilyinfiltrate the entire thickness of the fibrillated collagen material ina reasonable amount of time. Once the oil/solvent composition haspenetrated the entire thickness of the material, the solvent may then beremoved. Suitable oils and lubricants may include but are not limited tocastor oil, pine oil, lanolin, mink oil, neatsfoot oil, fish oil, sheabutter, aloe, and so forth.

Lubricating the dehydrated and crosslinked fibrillated collagen networkor hydrogel to form a leather material may result in a material havingproperties that are similar, or better, than the properties of naturalleather. The solutions that included a combination of oils and organicsolvent increased the mass and the softness (inversely proportional tothe slope of the stress-strain curve) of the dehydrated fibrillatedcollagen material. This is due to the combination of oils and organicsolvents penetrating the dehydrated fibrillated collagen material andonce penetrated through, the oils remained distributed throughout thematerial, while the organic solvents are able to evaporate away. Whilenot shown, the use of oils alone may not be as effective in penetratingentirely through the dehydrated fibrillated collagen material.

The resulting fibrillated collagen materials then may be treatedsimilarly to natural leather derived from animal hide or skin, and bere-tanned, dyed, and/or finished. Additional processing steps mayinclude: crosslinking, re-tanning, and surface coating. Crosslinking andre-tanning may include sub-processes such as wetting back (re-hydratingsemi-processed leather), sammying (45-55% water is squeezed from theleather), splitting (leather is split into one or more layers), shaving(leather is thinned), neutralization (pH of leather is adjusted tobetween 4.5 and 6.5), dyeing (leather is colored), fat liquoring (fats,oils, waxes are fixed to the leather fibers), filling (dense/heavychemicals to make leather harder and heavier), stuffing (fats, oils,waxes added between leather fibers), fixation (unbound chemicals arebonded/trapped and removed), setting (grain flatness are imparted andexcess water removed), drying (leather is dried to desired moisturelevels, 10-25%), conditioning (moisture is added to leather to a 18-28%level), softening (physical softening of leather by separating thefibers), or buffing (abrading surface of leather to reduce nap and graindefects). Surface coating may include any one or combination of thefollowing steps: oiling (leather coated with raw oil or oils), buffing,spraying, roller coating, curtain coating, polishing, plating,embossing, ironing, or glazing.

Unlike animal hides, where the hide has to be trimmed to obtain thedesired thickness or dimensions, the engineered leather material may befabricated with a wide range of thicknesses as well as the desireddimensions with a particular end product in mind.

The production of such engineered leather materials may also generateless waste by bypassing the step of removing excess proteins, fats, andhair necessary for treating natural animal hide in the leatherproduction process, which results in less environmental impact from thedisclosed process and the products derived from these methods.

The biofabricated materials disclosed herein are advantageouslycombined, incorporated or attached to other materials to form usefulcomposites. For example, a biofabricated coating may be applied to asecondary material such as a woven or nonwoven fabric or a plastic meshby dipping or spraying components forming the biofabricated material. Abiofabricated material may be incorporated on or laminated to one orboth sides of a flat secondary material. Specific embodiments of thesecomposite materials are described below.

Embodiments Composites

The invention includes, but is not limited to biofabricated materialscomponents having the features described below. The composites of theinvention include those where (i) one or more secondary components, suchas a particle, wire, fabric, or three dimensional object is incorporatedor embedded in a network of collagen fibrils, (ii) where a biofabricatedmaterial is coated or deposited, for example by filtration, on one sideof one or more secondary components such as a woven or nonwoven fabric,such as fabric, paper or regenerated cellulose, (iii) where abiofabricated component is coated or deposited on both sides of one ormore secondary materials having top and bottom sides or inner and outersides, or (iv) where a biofabricated material component and one or moresecondary components are adhered, attached or laminated to each other,for example, by direct lamination with or without an adhesive.

The biofabricated material once produced may be associated with the oneor more secondary components to form a composite. A composite may beformed simultaneously with the biofabricated material, for example, asecondary component such as a particle or fiber may be mixed withprecursors of a biofabricated material at any step in its production asdescribed herein. For example, a particulate or fibrous secondarymaterial can be mixed with collagen, collagen fibrils, crosslinkedcollagen fibrils, lubricated collagen fibrils, dehydrated collagenfibrils (including in powdered form), crosslinked, dehydrated andlubricated collagen fibrils, which are subsequently processed, alongwith the secondary material into a composite comprising thebiofabricated material component. The secondary component can be coatedwith or embedded in the resulting biofabricated material. An example ofthis is the deposit of crosslinked collagen fibrils on filter paper andthe subsequent dehydration and lubrication of the composite of thefilter paper (a secondary component) and the biofabricated materialdeposited by filtration on one side of the paper. Precursors of thebiofabricated material component may be coated or otherwise applied tosurface(s) of a secondary component and then processed into a finalbiofabricated material, for example, by at least one of fibrillation ofcollagen, crosslinking collagen fibrils, dehydration of collagen fibrilsor crosslinked collagen fibrils, and lubrication of collagen fibrils orcrosslinked collagen fibrils.

Alternatively, a biofabricated material component once produced, may becoated or laminated on at least one surface of a secondary componenthaving a top and bottom surface or inner and outer surface. In someembodiments, one or more layers of a flat secondary material will besandwiched between two layers of a biofabricated component which willform the external layers of a composite having the aesthetic qualitiesof the biofabricated component and strength, thickness or otherproperties conferred by the internally sandwiched secondary component.

The composites of the invention may also contain layered structures,including alternating or a repeating series of one or more layers of thebiofabricated and secondary components. These layers may appear in anyorder in a composite. Secondary component layers may be adjacent to eachother or to biofabricated layers. Biofabricated layers may be adjacentto each other or to layers of one or more secondary components. Suchcomposites may comprise adjacent or multiple layers of the biofabricatedcomponent with or without a non-collagenous secondary component. Forexample, multiple layers of a biofabricated component may be depositedon one side of a filter paper or mesh to increase the thickness of thebiofabricated material content of a composite.

The composites of the invention include, but are not limited to (i)those that involve dispersing, encapsulating, incorporating, depositing,or otherwise introducing at least one biofabricated material into oronto at least one porous, permeable, or absorptive secondary component;(ii) those that involve layering, laminating, depositing, coating orotherwise contacting at least one secondary component with at least onebiofabricated material; or (iii) those that sandwiching, layering,laminating, coating, or otherwise covering a top and bottom surface orinner and outer surface of at least one secondary component with atleast one biofabricated material.

They can involve incorporating or embedding one or more secondarymaterials into a network of collagen fibrils, for example, by mixing thesecondary materials with biofabricated material precursors or addingthem during the preparation of a biofabricated material comprising anetwork of crosslinked collagen fibrils. Examples of such secondarymaterials that may be incorporated into a biofabricated material toproduce a composite include particles, wires, fabrics, or threedimensional objects. Once the secondary component is incorporated into aprecursor of the biofabricated material component, the mixture may thenbe further processed into a biofabricated material that embeds,encapsulates or incorporates the secondary material.

These methods include coating or depositing a biofabricated material ora precursor of a biofabricated material, such as unfibrillated collagen,not crosslinked collagen fibrils, not dehydrated collagen fibrils or notlubricated collagen fibrils on a secondary component substrate, such asa woven or nonwoven fabric, paper, or regenerated cellulose. Forexample, depositing may be accomplished by filtering a solution orsuspension of collagen fibrils or crosslinked collagen fibrils throughsecondary material that retains the collagen fibrils on one side, forexample, filter paper. The deposited collagen fibrils may then befurther processed into a biofabricated material that is incorporatedinto or on one side of the secondary material. In some embodiments, thematerial may be deposited on both sides of a substrate. In others twosubstrates each containing a layer of biofabricated material can belaminated together with the biofabricated material facing inward oroutward. Preferably for the purpose of providing a leather-likeaesthetic, the layers of biofabricated material will face outward.

Biofabricated materials may be deposited or coated on two sides of asecondary material substrate to provide a leather-like aesthetic to theoutward facing sides. Alternatively, the biofabricated material can formone or more inner layers of a composite with the secondary materialfacing outward.

A composite material may be produced by attaching a biofabricatedmaterial once produced to one or more secondary components, for example,by coating or laminating the biofabricated material to at least onesurface of a secondary component having a top and bottom surface orinner and outer surface.

In some embodiments, a composite will be produced by sandwiching one ormore layers of a flat secondary material between at least two externallayers of a biofabricated component thus providing the aestheticqualities of the biofabricated component and strength, thickness orother properties conferred by the internally sandwiched secondarycomponent.

The composites of the invention may be produced by alternating orrepeating series of one or more layers of the biofabricated andsecondary components. These layers may appear in any order in acomposite. The method may comprise arranging secondary component layersadjacent to each other or to biofabricated layers. Biofabricated layersmay be adjacent to each other or to layers of one or more secondarycomponents. Such composites may comprise adjacent or multiple layers ofthe biofabricated component with or without a non-collagenous secondarycomponent. For example, multiple layers of a biofabricated component maybe deposited on one side of a filter paper or mesh to increase thethickness of the biofabricated material content of a composite.

Specific embodiments of the composite materials of invention include,without limitation, the following.

1. A composite material comprising:

(i) at least one porous, permeable, or absorptive secondary component,and

at least one biofabricated material comprising a network of non-humancollagen fibrils, wherein less than 10% by weight of the collagenfibrils in the material are in the form of collagen fibers having adiameter of 5 μm or more, in the form of fibrils aligned for 100 μm ormore of their lengths, or both; wherein said material contains no morethan 40% by weight water; and wherein said material contains at least 1%of a lubricant; or

(i) at least one porous, permeable, or absorptive secondary component,and

at least one biofabricated material comprising a network of recombinantnon-human collagen fibrils, wherein the collagen contains substantiallyno 3-hydroxyproline, and optionally, substantially no hydroxylysine;wherein said material contains no more than 25% by weight water; andwherein said material contains at least 1% of a lubricant; or

(ii) at least one layer of a secondary component, and

at least one layer of a biofabricated material comprising a network ofnon-human

collagen fibrils, wherein less than 10% by weight of the collagenfibrils in the material are in the form of collagen fibers having adiameter of 5 μm or more, in the form of fibrils aligned for 100 μm ormore of their lengths, or both; wherein said material contains no morethan 40% by weight water; and wherein said material contains at least 1%of a lubricant.;

(iii) at least one layer of a secondary component, and

at least one biofabricated material comprising a network of recombinantnon-human collagen fibrils, wherein the collagen contains substantiallyno 3-hydroxyproline, and optionally, substantially no hydroxylysine;wherein said material contains no more than 25% by weight water; andwherein said material contains at least 1% of a lubricant; or

(iv) at least one layer of a secondary component, and

at least two external layers of at least one biofabricated materialhaving a top and bottom surface, or inner and outer surface, comprisinga network of non-human collagen fibrils, wherein less than 10% by weightof the collagen fibrils in the material are in the form of collagenfibers having a diameter of 5 μm or more, in the form of fibrils alignedfor 100 μm or more of their lengths, or both; wherein said materialcontains no more than 40% by weight water; and wherein said materialcontains at least 1% of a lubricant; or

(v) at least one layer of a secondary component, and

at least two external layers of at least one biofabricated materialhaving a top and bottom surface, or inner and outer surface, comprisinga network of recombinant non-human collagen fibrils, wherein thecollagen contains substantially no 3-hydroxyproline, and optionally,substantially no hydroxylysine; wherein said material contains no morethan 25% by weight water; and wherein said material contains at least 1%of a lubricant.

2. A composite material comprising:

at least one porous, permeable, or absorptive secondary component, and

at least one biofabricated material, comprising a network of recombinantnon-human collagen fibrils, wherein the collagen contains substantiallyno 3-hydroxyproline, and optionally, substantially no hydroxylysine;wherein said material contains no more than 25% by weight water; andwherein said material contains at least 1% of a lubricant.

3. The composite according to embodiment 2, wherein the at least onebiofabricated material has been uniformly dispersed, encapsulated,incorporated, or otherwise introduced into the at least one secondarymaterial such that the concentration of the biofabricated material inidentical unit volumes of the composite varies by no more than 20%.

4. The composite according to embodiment 2, wherein the at least onesecondary material has been dispersed, encapsulated, incorporated, orotherwise introduced into a continuous phase of collagen fibrilscomprised by the at least one biofabricated material such that theconcentration of the secondary material in identical unit volumes of thecomposite varies by no more than 20%.

5. The composite according to embodiment 2, which has a top and bottomsurface or an inner and outer surface.

6. The composite according to embodiment 5, wherein the biofabricatedmaterial is only on or incorporated into one of the top, bottom, inneror outer surfaces.

7. The composite according to embodiment 5, wherein the secondarycomponent is a paper, fabric, or other nonwoven or woven fibrousmaterial.

8. The composite according to embodiment 5, wherein the biofabricatedmaterial is on or incorporated into both the top and bottom surfaces orboth the inner or outer surfaces.

9. The composite according to embodiment 2, wherein the secondarycomponent comprises at least one resin, polymer, or plastic.

10. The composite according to embodiment 2, wherein the secondarycomponent comprises at least one particle, bead, fiber, wire, mesh,woven, or nonwoven.

11. The composite according to embodiment 2, wherein the biofabricatedmaterial contains less than 1% by weight of actin, keratin, elastin,fibrin, albumin, globulin, mucin, mucinoids, noncollagen structuralproteins, and/or noncollagen nonstructural proteins;

12. The composite according to embodiment 2, wherein the biofabricatedmaterial is substantially free of non-collagenous proteins,carbohydrates, nucleic acids, or lipids, or immunogens, antigens, orallergens found in leather.

13. The composite according to embodiment 2, wherein the biofabricatedmaterial comprises at least 1% of at least one crosslinker.

14. The composite according to embodiment 2, wherein the diameters offibrils in the biofabricated material exhibit a substantially unimodaldistribution wherein at least 70% of the diameters of the fibrils in thematerial distribute around a single mode of diameter.

15. The composite according to embodiment 2, wherein the biofabricatedmaterial comprises at least one lubricant is selected from the groupconsisting of at least one fat, biological, mineral or synthetic oil,sulfonated oil, polymer, and organofunctional siloxane.

16. The composite of embodiment 1, wherein the biofabricated materialcomprises at least one photoluminescent agent.

17. The composite according to embodiment 2, wherein the biofabricatedmaterial comprises at least one electrically conductive or magneticagent.

18. The composite according to embodiment 2, wherein the biofabricatedmaterial has an elastic modulus between 100 kPa and 1,000 MPa, whereinthe elastic modulus varies by no more than 20% when measured at rightangles across identical lengths of the material and that has a tensilestrength of ranging from 1 MPa to 100 MPa, wherein the tensile strengthvaries by no more than 20% when measured at right angles acrossidentical lengths of the material.

19. The composite according to embodiment 2, wherein the biofabricatedmaterial further comprises a surface coating or surface finish; whereinthe surface coating or surface finish is distributed uniformlythroughout the material such that its concentration by weight in or onidentical unit volumes of the material varies by no more than 20%.

20. The composite according to embodiment 2, wherein the biofabricatedmaterial further comprises a dye, stain, resin, polymer, pigment orpaint, wherein the dye, stain, resin, pigment or paint is distributeduniformly throughout the material such that its concentration by weightin or on identical unit volumes of the material varies by no more than20%.

21. The composite according to embodiment 2, wherein the biofabricatedmaterial further comprises at least one filler, wherein the filler isdistributed uniformly throughout the material such that itsconcentration by weight in or on identical unit volumes of the materialvaries by no more than 20%.

Method of Making a Composite

Specific embodiments of a method for making a composite according to theinvention include, without limitation the following:

1. A method for making a composite material comprising:

(i) dispersing, encapsulating, incorporating, depositing, or otherwiseintroducing at least one biofabricated material into or onto at leastone porous, permeable, or absorptive secondary component; wherein the atleast one biofabricated material comprises a network of non-humancollagen fibrils, wherein less than 10% by weight of the collagenfibrils in the material are in the form of collagen fibers having adiameter of 5 μm or more, in the form of fibrils aligned for 100 μm ormore of their lengths, or both; wherein said material contains no morethan 40% by weight water; and wherein said material contains at least 1%of a lubricant; or

(ii) dispersing, encapsulating, incorporating, depositing, or otherwiseintroducing at least one biofabricated material into or onto at leastone porous, permeable, or absorptive secondary component; wherein saidand at least one biofabricated material comprises a network ofrecombinant non-human collagen fibrils, wherein the collagen containssubstantially no 3-hydroxyproline, and optionally, substantially nohydroxylysine; wherein said material contains no more than 25% by weightwater; and wherein said material contains at least 1% of a lubricant; or

(iii) layering, laminating, depositing, coating or otherwise contactingat least one secondary component, which has a top and bottom surface oran inner and outer surface, with at least one biofabricated materialthat comprises a network of non-human collagen fibrils, wherein lessthan 10% by weight of the collagen fibrils in the material are in theform of collagen fibers having a diameter of 5 μm or more, in the formof fibrils aligned for 100 μm or more of their lengths, or both; whereinsaid material contains no more than 40% by weight water; and whereinsaid material contains at least 1% of a lubricant; or

(iv) layering, laminating, depositing, coating or otherwise contactingat least one secondary component, which has a top and bottom surface oran inner and outer surface, with at least one biofabricated materialthat comprises a network of recombinant non-human collagen fibrils,wherein the collagen contains substantially no 3-hydroxyproline, andoptionally, substantially no hydroxylysine; wherein said materialcontains no more than 25% by weight water; and wherein said materialcontains at least 1% of a lubricant; or

(v) sandwiching, layering, laminating, coating, or otherwise covering atop and bottom surface or an inner and outer surface of at least onesecondary component with at least one biofabricated material thatcomprises a network of non-human collagen fibrils, wherein less than 10%by weight of the collagen fibrils in the material are in the form ofcollagen fibers having a diameter of 5 μm or more, in the form offibrils aligned for 100 μm or more of their lengths, or both; whereinsaid material contains no more than 40% by weight water; and whereinsaid material contains at least 1% of a lubricant; or

(vi) sandwiching, layering, laminating, coating, or otherwise covering atop and bottom surface or inner and outer surface of at least onesecondary component with at least one biofabricated material thatcomprises a network of recombinant non-human collagen fibrils, whereinthe collagen contains substantially no 3-hydroxyproline, and optionally,substantially no hydroxylysine; wherein said material contains no morethan 25% by weight water; and wherein said material contains at least 1%of a lubricant.

2. The method according to embodiment 1, wherein said method is (i) andwherein the at least one biofabricated material is produced by a processcomprising in any order:

fibrillating an aqueous solution or suspension of non-human collagenmolecules into collagen fibrils,

crosslinking said collagen fibrils by contacting them with at least onecrosslinking agent,

dehydrating the crosslinked collagen fibrils so that they contain lessthan 40% by weight water,

lubricating by incorporating at least 1% by weight of at least onelubricant into said material.

3. The method according to embodiment 2, wherein said biofabricatedmaterial is produced by fibrillating recombinant collagen.

4. The method according to embodiment 1, wherein said method is (ii) andwherein the at least one biofabricated material is produced by a processcomprising in any order:

fibrillating an aqueous solution or suspension of recombinant non-humancollagen molecules into collagen fibrils,

crosslinking said collagen fibrils by contacting them with at least onecrosslinking agent,

dehydrating the crosslinked collagen fibrils so that they contain lessthan 25% by weight water,

lubricating by incorporating at least 1% by weight of at least onelubricant into said material.

5. The method according to embodiment 4, wherein said fibrillating,crosslinking, dehydrating and/or lubricating is performed for a time andunder conditions that produce less than 10% by weight of the collagenfibrils in the biofabricated material in the form of collagen fibershaving a diameter of 5 μm or more, in the form of fibrils aligned for100 μm or more of their lengths, or both.

6. The method according to embodiment 1, wherein said method is (i) or(ii) and wherein the biofabricated material is incorporated into or ontothe at least one porous, permeable, or absorptive secondary component.

7. The method according to embodiment 1, wherein the secondary componentcomprises at least one resin, polymer, or plastic.

8. The method according to embodiment 1, wherein the secondary componentcomprises at least one fiber, bead, wire, particle, mesh, woven, ornonwoven.

9. The method according to embodiment 1, wherein the secondary componentcomprises at least one electrically conductive material, magneticmaterial, fluorescent material, bioluminescent material, phosphorescentmaterial, or combinations thereof.

10. The method of embodiment 1, wherein the biofabricated material isproduced by fibrillating non-human collagen molecules to produce fibrilsby at least one of adjusting a salt concentration or adjusting a pH ofan aqueous solution containing said collagen molecules.

11. The method of embodiment 1, wherein the biofabricated material isproduced by crosslinking collagen fibrils by contacting them with atleast one compound selected from the group consisting of an amine,carboxylic acid, sulfate, sulfite, sulfonate, aldehyde, hydrazide,sulfhydryl, diazirine, aryl, azide, acrylate, epoxide, phenol, chromiumcompound, vegetable tannin, and syntan.

12. The method according to embodiment 1, wherein the biofabricatedmaterial is produced by dehydrating the network of collagen fibrils bycontacting them with an agent that removes bound water from collagen.

13. The method according to embodiment 1, comprising lubricating thecollagen fibrils with least one lubricant selected from the groupconsisting of fat, biological, mineral or synthetic oil, cod oil,sulfonated oil, polymer, and organofunctional siloxane.

14. The method according to embodiment 1, wherein the biofabricatedmaterial is produced by uniformly distributing the lubricant on orthroughout the biofabricated material such that the concentration of thelubricant in identical unit volumes of the material varies by no morethan 20%.

15. The method according to embodiment 1, wherein the biofabricatedmaterial is produced by uniformly distributing a dye, stain, pigment,resin, polymer, or paint in or on it, wherein the concentration of thedye, stain, pigment, resin, polymer, or paint in identical unit volumesof the biofabricated material varies by no more than 20%.

16. The method according to embodiment 1, wherein the biofabricatedmaterial is produced by incorporating at least one filler into it.

Biofabricated Component of Composites

In one embodiment, the biofabricated material component comprises anetwork of collagen fibers, such as a biofabricated material orbiofabricated leather:

(i) comprising a network of non-human collagen fibrils,

wherein less than 5, 10, 15, 20, 25, 30, 35, or 40% by weight of thecollagen fibrils in the material are in the form of collagen fibershaving a diameter of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more and/or arein the form of fibrils aligned for 100 μm or more of their lengths;wherein said material contains no more than 10, 20, 30, 40, 50, or 60%by weight water; wherein said material contains at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, or 40% by weight of a lubricant; and whereinoptionally, the material comprises a top and bottom surface or an innerand outer surface; or

(ii) comprising a network of recombinant non-human collagen fibrils,wherein the collagen contains substantially no 3-hydroxyproline, andoptionally, substantially no hydroxylysine; wherein said materialcontains no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60%by weight water; wherein the material contains at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, or 40% of a lubricant; and wherein optionally,the material comprises a top and bottom surface or an inner and outersurface. Water content in this material is preferably no more than 25 to40%. Lubricant content may be selected to match or not exceed theabsorptive capacity of the biofabricated material for a lubricant. Sucha material may comprise mammalian collagen, such as bovine Type I orType III collagen. Preferably it will not contain hair, hairfollicle(s), or fat(s) of an animal that naturally expresses thecollagen molecules it contains. For example, it may contain less than 1,2, 3, 4, 5, 6, 7, 8, 9, or 10% by weight of actin, keratin, elastin,fibrin, albumin, globulin, mucin, mucinoids, noncollagen structuralproteins, and/or noncollagen nonstructural proteins found inconventional leather. It may be substantially free of other collagenousproteins, carbohydrates, nucleic acids, or lipids, or immunogens,antigens, or allergens found in a conventional leather, such as ananimal that naturally expresses the collagen molecules in abiofabricated material. Alternative embodiments may incorporate 1, 2, 3,4, 5, 6, 7, 8, 9, or 10% of one or more of actin, keratin, elastin,fibrin, albumin, globulin, mucin, mucinoids, noncollagen structuralproteins, and/or noncollagen nonstructural proteins found inconventional leather.

The collagen used to produce the fibrils in this material may beisolated from a natural source, preferably in a purified form, or it maybe recombinantly produced or produced by chemical synthesis. Collagengenerally contains 4-hydroxyproline. It may different in chemicalstructure from collagen obtained from a natural source, for example, ifmay contain a lower content of, or substantially no 3-hydroxyproline,and optionally, substantially no hydroxylysine, glycosylated orcrosslinked amino acid residues, or other post-translationalmodifications of a collagen amino acid sequence. Alternatively, it maycontain a higher content of hydroxylated amino acid residues,glycosylated residues, crosslinks or other chemical modifications.

The biofabricated material component described above generally comprisesa network of collagen fibrils which may exhibit a fibril density ofbetween 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, and1,000 mg/cc, preferably between 100 and 500 mg/cc. These fibrils ornetwork of fibrils can confer a grain texture, such as a top graintexture, feel, or aesthetic on a biofabricated material or biofabricatedleather. However, a biofabricated material can exhibit a porosity andother physical properties that are more uniform than a correspondingconventional leather which can be controlled or tuned by control ofcomposition, fibril size, crosslinking and lubricating in abiofabricated product.

In many embodiments, the biofabricated material component of a complexdescribed above will have a top and bottom surface, or an inner andouter surface, comprising the collagen fibrils. One or more of thesesurfaces may be externally exposed. A single layer of biofabricatedmaterial can exhibit substantially identical grain and appearance onboth of its sides, unlike conventional leather products where collagenfibril or fiber diameters increase for more inner layers of a hide.

In other embodiments a biofabricated material component of a complex maybe cast, molded or otherwise configured into a particular shape whichcan exhibit substantially uniform properties over its surface(s).

The collagen fibrils in the biofabricated material component of acomplex can be tuned to have a particular diameter. The distribution offibril diameters may exhibit a substantially unimodal distribution, abimodal distribution, a trimodal distribution or other multimodaldistributions. Multimodal distributions may be composed of two or moredifferent preparations of fibrils produced using different fibrillationconditions. In a substantially unimodal distribution >50, 60, 70, 80,90, 95 or 99% of diameters of the fibrils distribute around a singlemode. In bimodal distributions at least 5, 10, 15, 20, 25, 30, 35, 40,45, or 50% of the fibrils will distribute around one mode. In trimodaland other multimodal distributions, generally, at least about 5, 10, 15,20, 25, 30% or more (depending on the number of modes) of the fibrildiameters will distribute around a mode.

A biofabricated material component may contain fibrils where at least50, 60, 70, 80, 90, 95, or 99% of the collagen fibrils have diametersbetween 1 nm and 1 Fibril diameters may be determined by methods knownin the art including by visual inspection of micrographs or electronmicrographs, such as scanning or transmission electron micrographs. Forexample, the collagen fibrils may have a collective average orindividual fibril diameter ranging from 1, 2, 3, 4, 5, 10, 15, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or1,000 nm (1 μm).

The collagen fibrils in the biofabricated material component describedabove are usually crosslinked by contact with at least one agent thatforms crosslinks between collagen fibrils. Such a crosslinker may beselected from one or more of an amine, carboxylic acid, sulfate,sulfite, sulfonate, aldehyde, hydrazide, sulfhydryl, diazirine, aryl,azide, acrylate, epoxide, phenol, chromium compound, vegetable tannin,and syntan.

Crosslinking may be performed at a crosslinker concentration rangingfrom 1, 5, 10, 25, 50, 75 to 100 mM and may be conducted underconditions that uniformly expose collagen fibrils to the crosslinker sothat the average number of crosslinks formed is uniform and varies by nomore than 5, 10, 15, 20, 25, 30, 40, 45, or 50% in identical unitvolumes of the material.

A biofabricated material component may contain at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10% of a crosslinking agent based on the weight of thematerial or based on the weight of the collagen or collagen fibrils inthe material. The crosslinker may be present in a covalently ornon-covalently form, for example, it may be covalently bound to thecollagen fibrils. A crosslinker may be uniformly present in thebiofabricated material where its concentration by weight (or by mole)varies by no more than 5, 10, 15, 20, 25, 30, 40, 45, or 50% inidentical unit volumes of the material.

The biofabricated material or biofabricated leather component of acomplex described above contains a lubricant. Not lubricated materialscontaining a network of collagen fibrils can be produced, such aprecursor substrates for later lubrication, but can lack the flexibleand other useful properties of a lubricated product. Lubricants may beincorporated in any amount that facilitates fibril movement or thatconfers leather-like properties such as flexibility, decrease inbrittleness, durability, strength, increase resistance to fracture ortearing, or water resistance. A lubricant content can range from about0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, and 60% by weight of the biofabricated leather.

Lubricants used in the biofabricated component of a complex include, butare not limited to fats, biological, mineral or synthetic oils, cod oil,sulfonated oil, polymers, resins, organofunctional siloxanes, and otheragents used for fatliquoring conventional leather; mixtures thereof.Other lubricants include surfactants, anionic surfactants, cationicsurfactants, cationic polymeric surfactants, anionic polymericsurfactants, amphiphilic polymers, fatty acids, modified fatty acids,nonionic hydrophilic polymers, nonionic hydrophobic polymers, polyacrylic acids, poly methacrylic, acrylics, natural rubbers, syntheticrubbers, resins, amphiphilic anionic polymer and copolymers, amphiphiliccationic polymer and copolymers and mixtures thereof as well asemulsions or suspensions of these in water, alcohol, ketones, and othersolvents.

Solutions or emulsions containing a lubricant may be employed aslubricants, for examples, resins and other hydrophobic lubricants may beapplied as emulsions or in solvents suitable for dissolving them. Suchsolutions may contain any amount of the lubricant suitable forapplication to or incorporation into a biofabricated leather. Forexample, they may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 70, 80, 90, 95, or 99% of a lubricant or thesame, or a corresponding amount to volume of other ingredients, such asat least one aqueous solvent, such as water, alcohols, such C₁-C₆alcohols, like ethanol, ketones, such as C₁-C₆ ketones, aldehydes, suchas C₁-C₆ aldehydes, waxes, surfactants, dispersants or other agents.Lubricants may be in various forms, such as O/W or W/O emulsions, inaqueous or hydrophobic solutions, in sprayable form, or other formssuitable for incorporation or application to a biofabricated material.

Lubricants can be distributed uniformly throughout a biofabricatedmaterial component such that the concentration of the lubricant inidentical unit volumes of the material varies by no more than 5, 10, 15,20, 35, 30, 40, or 50% and may be compounded or mixed into formssuitable for uniform application to or into a biofabricated material.

Some embodiments of a biofabricated material component, or a complexthat incorporates it along with a secondary component, will exhibit manyadvantageous properties similar to leather or new or superior propertiescompared to conventional leather.

A biofabricated material component or a complex containing it can havean elastic modulus of at least 100 kPa. It can range from 100 kPa to1,000 MPa as well as any intermediate value in this range, such as 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 MPa.

A biofabricated material component or a complex containing it canexhibit a uniform elasticity, wherein the elastic modulus varies by nomore than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% when measured atangles differing by 30, 60, or 90 degrees (or at other angles) acrossidentical lengths or widths (or volumes or fixed cross-sectional areas)of the material.

A biofabricated material component or a complex containing it may bestretchable and can be elongated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 to 300% of itslength in a relaxed state. This range includes all intermediate values.

In some embodiments, a biofabricated material component or a complexcontaining it can have a tensile strength of at least 1 kPa. It canrange from 1 kPa to 100 MPa as well as any intermediate value in thisrange, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400,500 kPa; 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 MPa. Someembodiments will exhibit a uniform tensile strength, wherein the tensilestrength varies by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or50% when measured at angles differing by 30, 60, or 90 degrees (or atother angles) across identical lengths or widths (or volumes or fixedcross-sectional areas) of the material.

Some biofabricated material components or complexes containing them mayexhibit tear strength or resistance of at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150 or 200% more than thatof a conventional top grain or other leather of the same thicknesscomprising the same type of collagen, e.g., bovine Type I or Type IIIcollagen, processed using the same crosslinker(s) or lubricants. Someembodiments will exhibit a uniform tear resistance which varies by nomore than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% when measured atangles differing by 30, 60, or 90 degrees (or at other angles) acrossidentical lengths or widths (or volumes or fixed cross-sectional areas)of the material. A biofabricated material may have a tear strengthranging from about 1 to 500 N, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 as well as anyintermediate tear strength within this range.

A biofabricated material component, or a composite containing it, mayhave a softness as determined by ISO 17235 of 2, 3, 4, 5, 6, 7, 8, 10,11, 12 mm or more. v. Some embodiments will exhibit a uniform softnesswhich varies by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or100% when measured in otherwise identical unit areas or volumes of thebiofabricated material.

In other embodiments, a biofabricated material component, or compositecontaining it, exhibits a customized thickness to provide top grain likeproducts without the requirement for corium backing. In some embodimentsthe material or composite will have a top and bottom surface or an innerand outer surface which have identical or substantially the same grain,grain texture, feel, and appearance. Other embodiments of abiofabricated material component or a complex incorporating it areembossed with a pattern, distressed, or printed, stained or painted.Other embodiments of the biofabricated material component or complexcontaining it have a surface coating or surface finish, which may bedistributed uniformly on or throughout the material such that itsconcentration by weight in identical unit volumes or over unit areas ofthe material varies by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45,or 50%. Some embodiments of the biofabricated material component orcomplex containing it may contain a dye, stain, resin, polymer, pigmentor paint, optionally, wherein the dye, stain, resin, polymer, pigment orpaint is distributed uniformly throughout the material such that itsconcentration by weight in identical unit volumes or on unit areas ofthe material or complex varies by no more than 5, 10, 15, 20, 25, 30,35, 40, 45, or 50%.

Certain embodiments of the biofabricated material component describedabove may contain fillers as well as other substances or componentsincorporated into the network of collagen fibrils. For example, someembodiments will contain a filler, such as at least one of polymericmicrosphere(s), bead(s), fiber(s), wire(s), or organic salt(s) as asecondary component. These can be selected so as to control theorganization of the dehydrated collagen fibril network by keeping thefibrils spaced apart during drying. A filler may be soluble under someconditions or otherwise in a form that permits it removal from abiofabricated material after drying or other processing.

Other embodiments include secondary components of at least one woven ornonwoven material incorporated into the network of collagen fibrils or anetwork of collagen fibers incorporated into the nonwoven or wovenmaterial.

In some embodiments the biofabricated material component or complexincorporating it will be incorporated into other products such asfootwear, clothing, sportswear, uniforms, wallets, watchbands,bracelets, luggage, upholstery, or furniture.

Method for Making Biofabricated Component

The method according to the invention includes, but is not limited to,the following embodiments of a method for making a biofabricatedmaterial component.

A method for making:

(i) a biofabricated material component comprising a network of non-humancollagen fibrils, wherein less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35 or 40% by weight of the collagen fibrils in the materialare in the form of collagen fibers having a diameter of 1, 2, 3, 4, 5,6, 7, 8, 9, 10 μm or more and/or are in the form of fibrils aligned for25, 50, 100, 150, 200, 250, 300, 350 or 400 μm or more of their lengths;wherein said material contains no more than 10, 15, 20, 25, 30, 35,40,45, 50, 55 or 60% by weight water; and wherein said material contains atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40% of a lubricant,comprising in any order: fibrillating an aqueous solution or suspensionof non-human collagen molecules into collagen fibrils, crosslinking saidcollagen fibrils by contacting them with at least one crosslinkingagent, dehydrating the crosslinked collagen fibrils so that they containless than 40% by weight water, incorporating at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, or 40% by weight of at least one lubricant intosaid material, and, optionally, casting, molding, or otherwise formingsaid material that comprises a top and bottom surface or an inner andouter surface; or

(ii) a biofabricated material component comprising a network ofrecombinant non-human collagen fibrils, wherein the collagen containssubstantially no 3-hydroxyproline, and optionally, substantially nohydroxylysine; _wherein said material contains no more than 10, 15, 20,25, 30, 35,40, 45, 50, 55 or 60% by weight water; and wherein saidmaterial contains at least 1% of a lubricant comprising in any order:fibrillating an aqueous solution or suspension of non-human collagenmolecules into collagen fibrils, crosslinking said collagen fibrils bycontacting them with at least one crosslinking agent, dehydrating thecrosslinked collagen fibrils so that they contain no more than 5, 10,15, 20 or 25% by weight water, and incorporating at least 1, 2, 3, 4, 5,10, 15, 20, 30, 40, or 50% by weight of at least one lubricant into saidmaterial, and, optionally, casting, molding, or otherwise forming saidmaterial that comprises a top and bottom surface or an inner and outersurface.

The collagen or collagenous material for use in this method may comprisemammalian collagen, such as bovine Type I, Type III collagen or theother types and sources of collagens or collagenous proteins describedherein. It may be obtained from a mammal or other animal or, in someembodiments expressed recombinantly by Escherichia coli, Bacillussubtilis, or another bacterium; by Pichia, Saccharomyces, or anotheryeast or fungi; by a plant cell; by an insect cell or by a mammaliancell.

Collagen for use in the methods disclosed herein may be isolated fromcells, such as those described above, that are cultured in vitro, suchas from cultured mammalian or animal cells. Alternatively, collagen orcollagenous proteins may be obtained by other means, such as by chemicalsynthesis. It may different in chemical structure from collagen obtainedfrom a natural source, for example, if may contain a lower content of,or substantially no hydroxylysine or 3-hydroxyproline, glycosylated orcrosslinked amino acid residues, or other post-translationalmodifications of a collagen amino acid sequence. Alternatively, it maycontain a higher content of hydroxylated amino acid residues,glycosylated residues, crosslinks or other chemical modifications.

Preferably a collagen will not contain hair, hair follicle(s), or fat(s)of an animal that naturally expresses the collagen molecules it containsas these can detract from its uniformity, strength and aestheticproperties. For example, it may contain less than 1, 2, 3, 4, 5, 6, 7,8, 9, or 10% by weight of actin, keratin, elastin, fibrin, albumin,globulin, mucin, mucinoids, noncollagen structural proteins, and/ornoncollagen nonstructural proteins found in conventional leather. It maybe substantially free of other collagenous proteins, carbohydrates,nucleic acids, or lipids, or immunogens, antigens, or allergens found ina conventional leather, such as an animal that naturally expresses thecollagen molecules in a biofabricated material.

In some embodiments a collagen or collagen-like material may be purifiedto substantial homogeneity or may have a degree of purity notinconsistent with its ability to form fibrils, for example, it maycontain 25, 30, 40, 50, 60, 70, 80, 90, 95 or 99% by weight collagenbased on its total protein content or based on its total weight.Mixtures of different types of collagen or collagens from differentbiological sources may be used in certain embodiments to balance thechemical and physical properties of collagen fibrils or to produce amixture of fibrils having complementary properties. Such mixtures maycontain 1, 5, 10, 25, 50, 75, 95, or 99% by weight of a first collagenand 99, 95, 90, 75, 50, 25, 10 or 1% by weight of a second, third, orsubsequent collagen component. These ranges include all intermediatevalues and ratios of collagens where the total collagen content of allcollagen components by weight is 100%.

The methods disclosed herein can provide a biofabricated materialcomponent having substantially uniformly distributed fibrils,crosslinked fibrils, dehydrated fibrils and/or lubricated fibrils. Forexample, the fibrils may be distributed throughout the material so thatthe concentration by weight (or by number or average numbers) of thecollagen fibrils in identical unit volumes of the material varies by nomore than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%.

In some embodiments the biofabricated material component will beproduced by staking the material after the crosslinking, dehydratingand/or lubricating.

In the embodiments described herein, a collagen solution or suspensionis fibrillated, for example, by adjusting a salt concentration of thesolution or suspension, by adjusting its pH, for example, raising the pHof an acidic solution of collagen, or both. In some embodiments,fibrillation may be facilitated by including a nucleating agent. Saltsused for fibrillation include but are not limited to phosphate salts andchloride salts, such as Na₃PO₄, K₃PO₄, KCl, and NaCl. Salt concentrationduring fibrillation may be adjusted to range from 10 mM to 2M, or pH maybe adjusted to pH 5.5, 6.0, 6.5, 7.0, 8.0 or more with an acid, a base,or a buffer. Salt concentration and pH may be simultaneously adjusted toinduce or promote fibrillation. In certain embodiments of the methodsdescribed herein an aqueous solution or suspension of collagen moleculeshaving a pH below pH 6.0 can be fibrillated by adjusting the pH to pH6.0 to 8.0.

In some embodiments of the methods described herein, the collagenfibrils will be crosslinked during a process of their formation or aftercompletion of fibrillation. Crosslinking may be performed concurrentlywith incorporation of a secondary component.

In other embodiments, collagen fibrils are crosslinked by contactingthem with at least one amine, carboxylic acid, sulfate, sulfite,sulfonate, aldehyde, hydrazide, sulfhydryl, diazirine, aryl, azide,acrylate, epoxide, phenol, chromium compound, vegetable tannin, andsyntan.

One or more crosslinkers may be added at a concentration ranging from 1mM to 100 mM, for example at a concentration of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 6, 70, 75, 80,85, 90, 95or 100 mM.

The time, temperature and other chemical and physical conditions ofcrosslinking may be selected to provide a particular degree ofcrosslinking among the collagen fibrils so that the resultingcrosslinked fibrils contain a particular degree of one or more differentcrosslinkages. A resulting crosslinked fibril preparation may contain atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% or more of a crosslinking agentbased on the weight of the crosslinking agent and the weight of thecollagen or on the weight of a crosslinked network of collagen fibrils,such as a hydrogel. The crosslinker may be covalently- or non-covalentlybound to the collagen fibrils. The numbers of crosslinks between oramong collagen molecules, tropocollagen, or fibrils in identical unitvolumes of the material after crosslinking, or an average number ofcrosslinks between collagen molecules, tropocollagen, or collagenfibrils, may vary by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or50%.

The methods described herein require a dehydration or dewatering stepwhich may occur during fibrillation or crosslinking, or both, or afterfibrillation and crosslinking are substantially complete. These stepsmay be performed concurrently with incorporation of a secondarycomponent.

In some embodiments, dehydrating involves contacting a network ofcollagen fibrils with acetone, syntan, or other agent that removes boundwater from collagen. In other embodiments, some water may be removedfrom a fibril preparation or crosslinked fibril preparation byfiltration or evaporation and water remaining associated with thenetwork of collagen fibrils then removed using a solvent such as acetoneor other chemical agents that remove water.

The methods described herein generally require lubrication of thenetwork of collagen fibrils produced. Lubrication may take place duringfibrillation, crosslinking, of dehydration, or during any of thesesteps, or after one or more of these steps is substantially complete.Lubrication may be performed concurrently with incorporation of asecondary component.

In some embodiments lubrication will involve contacting a network ofcrosslinked collagen fibrils with one or more lubricants such as fats,biological, mineral or synthetic oils, cod oil, sulfonated oil,polymers, organofunctional siloxanes, and other agent used forfatliquoring conventional leather; or mixtures thereof.

In other embodiments, lubricant(s) will be applied using methods thatfacilitate uniform lubrication of a dehydrated crosslinked network ofcollagen fibrils, so that the concentration of the lubricant by weightin identical unit volumes of the material varies by no more than 5, 10,15, 20, 25, 30, 35, 40, 45, or 50%. Such application may occur bydip-coating, spray-coating, vapor deposition, spin-coating, Doctor Bladecoating, brush coating as well as other known coating or depositionmethods.

In further embodiments of the methods described herein, a surfacecoating or surface finish is applied to a biofabricated material. Whilethese may be applied to a surface of a material comprising a network ofcollagen fibrils during the various steps of the preparation of abiofabricated material, they will generally be applied to a crosslinked,dehydrated and lubricated product. The uniform lubrication made possibleby the methods described herein facilitates the successful uniformapplication and adherence of such coatings or finishes.

In other embodiments, the methods described herein can includeincorporating or contacting a biofabricated material during the varioussteps of its preparation or after it has been crosslinked, dehydratedand lubricated with other functional ingredients including, but notlimited to a dye, stain, pigment, resin, polymer, or paint. In furtherembodiments, these functional ingredients may be applied or incorporatedunder conditions that uniformly distribute these agents on or throughoutthe material so that their concentration by weight in identical unitvolumes of the material varies by no more than 5, 10, 15, 20, 25, 30,35, 40, 45, or 50%.

In other embodiments, the method described herein involves incorporatinga filler of secondary component into a biofabricated material during thevarious steps of its preparation or after it has been crosslinked,dehydrated and lubricated. Generally, these fillers are incorporatedprior to dehydration, for example, during fibrillation or crosslinking.Such fillers include, but are not limited to polymeric microspheres,beads, fibers, wires, or organic salts.

Some embodiments of the methods described above will involveincorporating into or onto a biofabricated material during or after itspreparation at least one woven or nonwoven material. For example, byfiltering crosslinked fibrils using a woven or nonwoven paper or fabricmaterial. Other embodiments involve incorporating a biofabricatedmaterial during or after its preparation into at least one woven ornonwoven material.

Commercial embodiments of the method involving incorporating abiofabricated material into products such as footwear, clothing,sportswear, uniforms, wallets, watchbands, bracelets, luggage,upholstery, furniture, or other industrial, commercial or consumerproducts.

The following non-limiting Examples are illustrative of the presentinvention. The scope of the invention is not limited to the detailsdescribed in these Examples.

EXAMPLE 1 Controlling the Thickness of Biofabricated Leather

The thickness of the biofabricated material used in a composite may becontrolled by adjusting collagen content. Hydrogels of extracted bovinetype I collagen were formed at different collagen concentrations andvolumes to produce dried collagen materials of different thicknesses.Collagen was dissolved in 0.01N HCl at either 5 g/L or 9 g/L, then 1part 10× PBS was added to 9 parts dissolved collagen to induce collagenfibrillation and gel formation.

Solutions of either 0.8 L or 1.6 L of the fibrillating collagen werethen cast into molds and incubated at 25° C. to allow hydrogelformation. The 0.8 L solution produced a gel of 1.5 cm thickness whilethe 1. 7L solution produced a gel of 3.0 cm thickness. These gels weredehydrated and lubricated in acetone, then dried and mechanically stakedinto a leather like material. The thickness of the final dried materialcorrelated with the total amount of collagen in the starting hydrogel.

The thickness of biofabricated leather was controlled by varying itstotal collagen content. Samples A, B and C were produced using 4, 7.2 or14.4 gr of collagen, respectively, in a volume (hydrated gel area) of525 cm². Biofabricated leathers were produced from each sample bycrosslinking, lubricating and dewatering As shown in Table 1, increasingthe content of collagen in the gels increased the thickness of theresulting biofabricated leather.

TABLE 1 Gel Gel Gel Total Leather Density Volume Thickness CollagenThickness Sample (g/L) (L) (cm) (g) (mm) A 5 0.8 1.5 4 0.1 B 9 0.8 1.57.2 0.2 C 9 1.6 3.0 14.4 1.1

EXAMPLE 2 Production of Biofabricated Leather from Type I Collagen

The biofabricated component of the composites described herein may beproduced from Type I collagen.

Type I collagen was purchased from Wuxi Biot Bio-technology Company,ltd. (Medical Collagen Sponge). The collagen was isolated from bovinetendon by acid treatment followed by pepsin digestion, and was purifiedby size exclusion chromatography, frozen and lyophilized.

The lyophilized protein (4.1 g) was dissolved in 733 ml 0.01 N HCL usingan overhead mixer. After the collagen was adequately dissolved, asevidenced by a lack of solid collagen sponge in the solution (at least 1hr mixing at 1,600 rpm), 82 uL of the tanning agent Relugan GTW wasadded to the solution followed by 81 mL of a 10× PBS, pH 11.2 to raisethe pH of the solution to 7.2.

The solution was then mixed for 3 min before pouring the solution into asilicon mold. The collagen solution was incubated in the silicon moldfor 2 hrs at 25° C. to allow the collagen to fibrillate into aviscoelastic hydrogel.

Plateau of rheological properties along with solution opacity (asmeasured by absorbance of 425 nm light) indicated that fibrillation wascomplete at this point and the presence of collagen fibrils wasconfirmed with scanning electron microscopy (FIG. 3) and transmissionelectron microscopy (FIG. 4).

The fibrillated collagen hydrogel was removed from the molds and placedin 700 mL of acetone in a plastic jar and shaken on an orbital shaker at40 rpm at 25° C. The hydrogel was dehydrated by refreshing the acetoneafter an overnight incubation followed by 5×1 hr washes and anotherovernight incubation. Acetone was refreshed after each wash to removewater from the gel.

Following acetone dehydration, the collagen gel was incubated in a fatliquor solution containing 20% (v/v) of either cod liver oil or castoroil in 80% acetone or ethanol, respectively, overnight while shaking at40 rpm.

Following incubation in the fat liquor solution, the collagen gel wasdried at 37 C. After drying, the material became soft and leather-likeor a biofabricated leather. Excess oil can be removed to improve theleather-like aesthetic of the materials.

Sample weights and mechanical analysis confirmed penetration of the oilsinto the fibrillar gel. By dissolving the oils in good solvents, theoils were able to penetrate the fibrillar collagen network as evidencedby an increase in dry weight of the materials as well as a decrease inthe elastic modulus of the material compared to hydrogels that we notdehydrated or fat liquored in solvent.

The biofabricated leather had a grain texture on both the top and bottomsurfaces and consistently absorbed dyes on both the top and bottomsurfaces.

EXAMPLE 3 Production of Biofabricated Leather from Type III Collagen

The biofabricated component of the composites described herein may beproduced using Type III collagen.

A solution of recombinant collagen type III at 2.5 mg/ml in 0.01 N HCl(FibroGen, Inc.) was fibrillated by adding 1 part of a 200 mM of sodiumphosphate solution (22 mL), pH 11.2 to 9 parts of the collagen solution(200 mL) to increase the pH to 7 and stirred 2 hours at roomtemperature.

Fibrillation was confirmed by measuring 400 nm absorbance of thesolution over time.

After fibrillation, the fibrils were tanned by adding Relugan GTW (2%w/w offer on the collagen) to the fibril suspension and mixing for 30min.

The tanned collagen fibrils were then centrifuged at 3,500 RPM for 30minutes to concentrate the fibrils to a concentration of 10 mg/ml. The10 mg/ml fibril pellet was further centrifuged using an ultra-centrifugeat 21,000 RPM for 30 minutes yielding a fibril gel with a concentrationof ˜40-50 mg/ml.

The physical properties of the fibril gel were assessed with a rheometer

The storage modulus and complex viscosity demonstrate a mostly elasticmaterial.

This fibril gel was then dried in a food dehydrator set to 37° C. for 18hrs.

After drying, the material was dyed and retanned by incubating in asolution of Lowepel acid black dye (2% w/w offer on the collagen) andLubritan WP (20% w/w offer on the collagen).

The material was drummed in this solution and squeezed to ensurepenetration of dye and syntan into the material. The material was thenfinally dried and staked to produce a leather-like material.

EXAMPLE 4 Production of Biofabricated Leather from Type III Collagen

The biofabricated component of the composites described herein may beproduced using Type II collagen.

Recombinant collagen type III was purchased from Fibrogen, Inc. Thecollagen was supplied at a concentration of 2.5 mg/mL in 0.01N HCl.

To initiate the assembly of collagen fibrils, 1 part 200 mM Na₂HPO₄, pH11.2 (100 mL) was added to 9 parts of the stock collagen type IIIsolution at room temperature to bring the solution to pH 7.2. Thesolution was mixed at 1600 rpm for 1 hr using an overhead mixer.

After 1 hr of stirring, the collagen fibrils were reacted with ReluganGTW which was added to the solution at a 2% (w/w) offer on the mass ofthe collagen. The solution was mixed at 1600 rpm for 1 hr using anoverhead mixer.

Lipoderm A1 and Tanigan FT were then added to the solution at offers of80% (w/w) each on the mass of the collagen. The solution was mixed at1,600 rpm for 30 min using an overhead mixer. The pH of the solution wasthen lowered to 4 using a 10% (v/v) formic acid solution. The solutionwas mixed at 1,600 rpm for 30 min using an overhead mixer.

144 mL of the solution was then filtered through a 47 mm Whatman no. 1membrane using a Buchner funnel attached to a vacuum pump (pressure of−27 in Hg) and a rubber darn on top of the Buchner funnel. Vacuum waspulled for 18 hrs.

The concentrated fibril tissue was then allowed to dry under ambientconditions and hand staked for 30 min by rolling, bending and pullingthe material to produce a leather-like material.

EXAMPLE 5 Expancell

Type I bovine collagen, isolated from bovine tendon by acid treatmentfollowed by pepsin digestion and purified by size exclusionchromatography, frozen and lyophilized, was purchased from Wuxi BiotBio-technology co., Ltd. (Medical Collagen Sponge).

Using an overhead mixer, 10 gr of the lyophilized collagen protein wasdissolved by mixing at 1,600 rpm in 1 L of 0.01N HCl, pH 2, for at leastone hour until no solid collagen sponge was present.

111.1 ml of 200 mM sodium phosphate (pH adjusted to 11.2 with sodiumhydroxide) was then added to raise the pH of the collagen solution to7.2.

The pH 7.2 collagen solution was then stirred for 10 minutes and 0.1 mlof a 20% Relugan GTW (BASF) as a crosslinker, which was 2% on the weightof collagen, was added to produce crosslinked collagen fibrils.

The crosslinked collagen fibrils were then mixed with 5 ml of 20%Tanigan FT (Lanxess) and stirred for one hour.

Subsequently, 1 gr of Expancel Microspheres 461 WE 20 d36 (AkzoNobel),which is 10% of the weight of the collagen) and 40 ml of Truposol Ben(Trampler), which is 80% of the weight of the collagen, were added andstirred for an additional hour using an overhead stirrer.

The pH of the solution was the reduced to pH 4.0 by addition of 10%formic acid and stirred for an hour.

After the reduction in pH, 150 ml of the solution was filtered through90 mm Whatman No. 1 membrane using a Buchner funnel attached to a vacuumpump at a pressure of −27 mmHg.

The concentrated fibril tissue was then allowed to dry under ambientconditions and hand staked for 30 minutes by rolling, bending andpulling the material to produce a leather-like material. This materialmay be incorporated into the composites described herein.

EXAMPLE 6 Titanium Dioxide (White Pigment)

Type I bovine collagen was purchased from Wuxi Biot Bio-technology co.,Ltd. (Medical Collagen Sponge). This source of collagen is type Icollagen isolated from bovine tendon by acid treatment followed bypepsin digestion and purified by size exclusion chromatography, frozenand lyophilized. The lyophilized protein (10 grams) was dissolved in 1 Lof 0.01N HCl, pH 2 using an overhead mixer. After the collagen wasadequately dissolved, as evidenced by a lack of solid collagen sponge inthe solution (at least 1 hr mixing at 1,600 rpm), 111.1 ml of 200millimolar sodium phosphate (pH adjusted to 11.2 with sodium hydroxide)to raise the pH of the solution to 7.2. The resulting collagen solutionwas stirred for 10 minutes and 0.1 ml of a 20% Relugan GTW (BASF)crosslinker solution, which was 2% on the weight of collagen.

To the crosslinked collagen fibril solution was added 5 mls of 20%Tanigan FT (Lanxess) was added followed by stirring for one hour.

Following Tanigan-FT addition, 1 gr Expancel Microspheres (10% on theweight of collagen) 461 WE 20 d36 (AkzoNobel), 40 mls (80% on the weightof collagen) of Truposol Ben (Trumpler) and 2 mls (10% on the weight ofcollagen) of PPE White HS a pa (Stahl) was added and stirred foradditional hour using an overhead stirrer.

The pH of the solution was reduced to 4.0 using 10% formic acid andstirred for an hour.

After pH change, 150 ml of the solution was filtered through 90 mMWhatman No. 1 membrane using a Buchner funnel attached to a vacuum pumpat a pressure of −27 mmHg.

The concentrated fibril tissue was then allowed to dry under ambientconditions and hand staked for 30 minutes by rolling, bending andpulling the material to produce a leather-like material. This materialmay be incorporated into the composites described herein.

EXAMPLE 7 Hycar Resin (26552)

Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd.(Medical Collagen Sponge). This source of collagen is type I collagenisolated from bovine tendon by acid treatment followed by pepsindigestion and purified by size exclusion chromatography, frozen andlyophilized.

The lyophilized protein (10 grams) was dissolved in 1 liter of 0.01NHCl, pH 2 using an overhead mixer. After the collagen was adequatelydissolved, as evidenced by a lack of solid collagen sponge in thesolution (at least 1 hr mixing at 1600 rpm), 111.1 ml of 200 mM sodiumphosphate (pH adjusted to 11.2 with sodium hydroxide) to raise the pH ofthe solution to 7.2.

The resulting collagen solution was stirred for 10 minutes and 0.1 ml ofa 20% Relugan GTW (BASF) crosslinker solution, which was 2% of theweight of the collagen, tanning agent solution was added.

To the crosslinked collagen fibril solution was added 5 mls of 20%Tanigan FT (Lanxess) was added and stirred for one hour. FollowingTanigan-FT addition, 1 gram Expancel Microspheres (10% on the weight ofcollagen) 461 WE 20 d36 (AkzoNobel) ,40 mis (80% on the weight ofcollagen) of Truposol Ben (Trumpler) and 2 mls (10% on the weight ofcollagen) of PPE White HS a pa (Stahl) was added and added and stirredfor additional hour using an overhead stirrer.

The pH of the solution was reduced to 4.0 using 10% formic acid and avariety of offers of Hycar Resin 26552 (Lubrizol) was added and stirredfor an additional hour. Following pH change and resin addition 150 ml ofthe solution was filtered through 90 millimeter Whatman No. 1 membraneusing a Buchner funnel attached to a vacuum pump at a pressure of −27mmHg. To facilitate activation, the Hycar Resin 26552 is mixed with thefibril solution and heated at 50° C. for 2 hrs.

The concentrated fibril tissue was then allowed to dry under ambientconditions and hand staked for 30 minutes by rolling, bending andpulling the material to produce a leather-like material. This materialmay be incorporated into the composites described herein.

The addition of resin lead to improved mechanical properties as shownbelow in FIG. 1.

After pH change, 150 ml of the solution was filtered through 90millimeter Whatman No. 1 membrane using a Buchner funnel attached to avacuum pump at a pressure of −27 mmHg. The solution immediately formed agreen precipitate and was unable to be filtered.

Example Substrates Crosslinker Dehydrator Lubricant Result 5 Type IRelugan Tanigan Truposol Leather-like collagen + GTW FT materialExpancel microspheres 6 ″ ″ ″ ″ Leather-like material 7 ″ ″ ″ ″Leather-like material, better mechanical properties

After Relugan is a retanning agent based on polymer, resin or aldehyde.Tanigan is a sulfone-based syntan. Truposol Ben is a fatliquor forchrome-free leather. Lipoderm Liquor A1 is a fatliquor based on longchain alcohol, paraffin, anionic surfactants, in water Hycar Resin26552: formaldehyde-free acrylic based emulsion.

EXAMPLE 8 Encapsulated Carbon Fibers

Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd.(Medical Collagen Sponge). This source of collagen is type I collagenisolated from bovine tendon by acid treatment followed by pepsindigestion and purified by size exclusion chromatography, frozen andlyophilized. The lyophilized protein (4.1 g) was dissolved in 733 mL of0.01N HCl, pH 2 using an overhead mixer. After the collagen wasadequately dissolved, as evidenced by a lack of solid collagen sponge inthe solution (at least 1 hr mixing at 1,600 rpm), 82 uL of the tanningagent Relugan GTW was added to the solution followed by 81 mL of a 10×PBS, pH 11.2 to raise to pH of the solution to 7.2. The solution wasmixed for 3 min, then poured into a mold containing a secondary materialof 0.25 inch chopped carbon fibers. Carbon fibers were purchased fromFibre Glast Developments Corp. The carbon fibers were mixed in thecollagen solution to disperse the fibers throughout the collagen matrix.The collagen solution was incubated in the silicon mold for 2 hrs at 25°C. to allow the collagen to fibrillate into a viscoelastic hydrogel,encapsulating the carbon fibers.

The fibrillated collagen hydrogels with encapsulated carbon fibers wereremoved from the molds and dehydrated in a series of acetone solutions(5×1 hr at 25° C., 40 rpm). Following acetone dehydration, the collagengel was incubated in a fat liquor solution containing 20% (v/v) codliver oil in 80% acetone overnight while shaking at 40 rpm. Followingincubation in the cod liver oil solution, the collagen gel was dried at37° C. The fibrillated collagen hydrogel was removed from the molds andplaced in 700 mL of acetone in a plastic jar and shaken on an orbitalshaker at 40 rpm at ^(25oC). The hydrogel was dehydrated by refreshingthe acetone after an overnight incubation followed by 5×1 hr washes andanother overnight incubation. Acetone was refreshed after each wash toremove water from the gel. Following acetone dehydration, the collagengel was incubated in a fat liquor solution containing 20% (v/v) ofeither cod liver oil or castor oil in 80% acetone or ethanol,respectively, overnight while shaking at 40 rpm. Following incubation inthe fat liquor solution, the collagen gel was dried at 37 C. Afterdrying, the material becomes soft and leather-like. Further, the carbonfibers are encapsulated within the tanned and fat liquored collagennetwork and can be handled without delaminating or pulling out of thebiofabricated leather.

EXAMPLE 9 Layered Non-Woven

Bovine collagen was dissolved as in Example 8. Once the collagen wasdissolved, as evidenced by a lack of solid collagen sponge in thesolution (at least 1 hr mixing at 1600 rpm), 0.2 g of Lowepel acid blackdye dissolved in 5 mL DI water was added dropwise to the stirringcollagen solution. The dye was mixed for 1 hr@1600 rpm to allow dyefixation to collagen. 82 uL of the tanning agent Relugan GTW was thenadded to the solution followed by 81 mL of a 10× PBS, pH 11.2 toincrease to pH of the solution to 7.2. The solution was mixed for 3 minand integrated with a secondary material of wool nonwoven felt using avacuum technique. Wool felts were purchased from US Felts and treatedwith 1M hydroxylamine, 1 g/L triton n-57 surfactant, pH 8 overnight@50°C. to remove surface lipids and increase wettability and reactivity ofthe wool fibers. 60 mL of the collagen precursor solution was pulledinto the wool felt under house vacuum. A gradient of dye was visiblefrom the top surface of the felt to the bottom. Following integrationwith the collagen solution, the wool felt was laid topside down onto afreshly cast collagen precursor solution. The collagen and wool felt wasincubated for 2 hrs@25° C. to allow fibrillation. After fibrillation,the material was dried in a dehydrator@37 C. The dried material wasstaked into a soft, leather-like material with wool backing.

EXAMPLE 10 Embedded Fabrics with Photoluminescent Patterns

Qdots functionalized with a primary amine and PEG spacer were purchasedfrom Sigma. The Qdots were diluted 1:10 in a collagen precursor solution(5 wt % col type I, 1×PBS, 0.02 uL GTW/mg col) chilled on ice. TheQdot/collagen solution was then screen printed onto a secondary materialof silk woven fabric in the shape of an “M”. The Qdot/collagen screenprinted fabric was incubated for 1 hr@RT before encapsulating the fabricin a collagen gel. As in Example 2, the collagen precursor solution (5mg/mL col type I, 1×PBS, 0.02 uL GTW/mg col) was cast into a siliconmold 3 min after adding the PBS and the fabric was placed in the middleof the collagen solution. The solution was incubated at 25° C. for 1 hrto allow fibrillation and then the gel with encapsulated fabric wasdehydrated in a series of acetone, followed by fat liquoring in codoil/acetone and drying. After drying and staking, the material wasexposed to a UV light source to illuminate the embedded Qdot “M”.

EXAMPLE 11 Embedded Three-Dimensional Objects

Qdots functionalized with a primary amine and PEG spacer were purchasedfrom Sigma. The Qdots were diluted 1:10 in a Slygard 184polydimethylsiloxane (PDMS) base followed by mixing the Qdot/base 10:1with a curing agent. After mixing, the Qdot/base/curing agent solutionwas cast into a mold in the shape of an “M”. The PDMS “M” was curedovernight at 40° C. then removed from the mold to produce an elastomericand photoluminescent “M”. As in Example 2, the collagen precursorsolution (5 mg/mL col type I, 1× PBS, 0.02 uL GTW/mg col) was cast intoa silicon mold 3 min after adding the PBS and the PDMS “M” was placed inthe middle of the collagen solution. The solution was incubated at 25°C. for 1 hr to allow fibrillation and then the gel with encapsulatedfabric was dehydrated in a series of acetone, followed by fat liquoringin cod oil/acetone and drying (see Example 2 for details). After dryingand staking, the encapsulated three-dimensional “M” produced a tactilepattern on the surface of the biofabricated leather in the shape of the“M”. In addition, the material was exposed to a UV light source toilluminate the embedded Qdots in thE PDMS “M”.

EXAMPLE 12 Wool Felt Composite

The process of Example 9 is repeated with wool felt and the collagenprecursor solution of Example 6. A composite leather is formed.

EXAMPLE 13 Lycra® Composite

A 3″ by 3″ sample of the leather of Example 2 is laminated with a 3″ by3″ polyester-polyurethane copolymer felt (Lycra®) with a holt meltadhesive at 50° C. A leather-secondary material backed composite isformed.

EXAMPLES 14-20

As shown by the Examples 14-20 below, the biofabricated material of theinvention can be successfully applied or integrated in to secondarycomponents to produce strong leather-like composites.

EXAMPLE 14 Spacer Fabric

Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd.(Medical Collagen Sponge). This source of collagen is type I collagenisolated from bovine tendon by acid treatment followed by pepsindigestion and purified by size exclusion chromatography, frozen andlyophilized. The lyophilized protein (10 grams) was dissolved in 1 litreof 0.01N HCl, pH 2 using an overhead mixer.

After the collagen was adequately dissolved, as evidenced by a lack ofsolid collagen sponge in the solution (at least 1 hr mixing at 1,600rpm), 111.1 millilitres of 200 millimolar sodium phosphate (pH adjustedto 11.2 with sodium hydroxide) to raise the pH of the solution to 7.2.The resulting collagen solution was stirred for 10 minutes and 0.1millilitres of a 20% Relugan GTW (BASF) (2% on the weight of collagen)tanning agent solution was added.

To the crosslinked collagen fibril solution was added 5 mls of 20%Tanigan FT (Lanxess) was added and stirred for one hour.

Following Tanigan-FT addition, 1 gram Expancel Microspheres (10% on theweight of collagen) 461 WE 20 d36 (AkzoNobel) and 40 mls (80% on theweight of collagen) of Truposol Ben (Trumpler) was added and added andstirred for additional hour using an overhead stirrer. The pH of thesolution was changed to 4.0 using 10% formic acid and stirred for anhour.

After pH change a 75 mm disc of a 100% polyester 3D spacer fabric wascut out and placed on top of a 90 mm Whatman no. 1 membrane, a thinlayer of high vacuum grease (Dow Corning) was applied around the rim ofthe membrane to hold down the material whilst filtering.

150 mL of the solution was then filtered through the textile and Whatmanno. 1 membrane using a Buchner funnel attached to a vacuum pump(pressure of −27 in Hg). Vacuum was pulled for 40 mins.

The concentrated fibril tissue was then allowed to dry in a humiditychamber at 20° C. at 65%. When the concentrated fibril tissue hadreached 20% moisture it was pressed in a carver press 50° C. for 10 minsat 1 metric tonne of pressure and hand staked for 30 min by rolling,bending and pulling the material to produce a leather-like material.

The spacer fabric remained integrated into the fibril tissue, resultingin a leather-like material that had an exposed fabric back on one sideand an embossed pattern on its surface created by the embedded textile.The material was finished with a high performance coating, routinelyused in the footwear industry.

EXAMPLE 15

The procedure of Example 2 was repeated substituting the 75 mm disc forsmaller sections that are zonally integrated into the end material.

EXAMPLE 16 Polyester Mesh Netting

Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd.(Medical Collagen Sponge). This source of collagen is type I collagenisolated from bovine tendon by acid treatment followed by pepsindigestion and purified by size exclusion chromatography, frozen andlyophilized. The lyophilized protein (10 grams) was dissolved in 1 litreof 0.01N HCl, pH 2 using an overhead mixer.

After the collagen was adequately dissolved, as evidenced by a lack ofsolid collagen sponge in the solution (at least 1 hr mixing at 1600rpm), 111.1 millilitres of 200 millimolar sodium phosphate (pH adjustedto 11.2 with sodium hydroxide) to raise the pH of the solution to 7.2.

The resulting collagen solution was stirred for 10 minutes and 0.1millilitres of a 20% Relugan GTW (BASF)(2% on the weight of collagen)tanning agent solution was added.

To the crosslinked collagen fibril solution was added 5 mls of 20%Tanigan FT (Lanxess) was added and stirred for one hour. FollowingTanigan-FT addition, 1 gram Expancel Microspheres (10% on the weight ofcollagen) 461 WE 20 d36 (AkzoNobel) and 40 mis (80% on the weight ofcollagen) of Truposol Ben (Trumpler) was added and added and stirred foradditional hour using an overhead stirrer. The pH of the solution waschanged to 4.0 using 10% formic acid and stirred for an hour.

After pH change a 75 mm disc of a polyester mesh netting was cut out andplaced on top of a 90 mm Whatman no. 1 membrane, a thin layer of highvacuum grease (Dow Corning) was applied around the rim of the membraneto hold down the material whilst filtering. 150 mL of the solution wasthen filtered through the textile and Whatman no. 1 membrane using aBuchner funnel attached to a vacuum pump (pressure of −27 in Hg). Vacuumwas pulled for 40 mins.

The concentrated fibril tissue was then allowed to dry in a humiditychamber at 20° C. at 65%.

When the concentrated fibril tissue had reached 20% moisture it waspressed in a carver press 50° C. for 10 mins at 1 metric tonne ofpressure and hand staked for 30 min by rolling, bending and pulling thematerial to produce a leather-like material.

The fabric was removed 15 mins into staking, resulting in a double-sidedgrain with a different textured surface, and aesthetic, on each side ofthe material. The material was finished with a high performance coating,routinely used in the footwear industry.

EXAMPLE 17 Polyester Textile

The procedure of Example 3 is repeated with the additional step oflaminating a 100% polyester technical textile to one side of thematerial.

EXAMPLE 18 Coating

Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd.(Medical Collagen Sponge). This source of collagen is type I collagenisolated from bovine tendon by acid treatment followed by pepsindigestion and purified by size exclusion chromatography, frozen andlyophilized. The lyophilized protein (10 grams) was dissolved in 1 litreof 0.01N HCl, pH 2 using an overhead mixer.

After the collagen was adequately dissolved, as evidenced by a lack ofsolid collagen sponge in the solution (at least 1 hr mixing at 1600rpm), 111.1 millilitres of 200 millimolar sodium phosphate (pH adjustedto 11.2 with sodium hydroxide) to raise the pH of the solution to 7.2.

The resulting collagen solution was stirred for 10 minutes and 0.1millilitres of a 20% Relugan GTW (BASF) (2% on the weight of collagen)tanning agent solution was added.

To the crosslinked collagen fibril solution was added 5 mls of 20%Tanigan FT (Lanxess) was added and stirred for one hour. FollowingTanigan-FT addition, 1 gram Expancel Microspheres (10% on the weight ofcollagen) 461 WE 20 d36 (AkzoNobel) and 40 mls (80% on the weight ofcollagen) of Truposol Ben (Trumpler) was added and added and stirred foradditional hour using an overhead stirrer. The pH of the solution waschanged to 4.0 using 10% formic acid and stirred for an hour.

After pH change 150 mL of the solution was then filtered through a 90 mmWhatman no. 1 membrane using a Buchner funnel attached to a vacuum pump(pressure of −27 in Hg). Vacuum was pulled for 40mins. The concentratedfibril tissue was then allowed to dry in a humidity chamber at 20° C. at65%.

When the concentrated fibril tissue had reached 20% moisture it waspressed in a carver press 50° C. for 10 mins at 1 metric tonne ofpressure and hand staked for 30 min by rolling, bending and pulling thematerial to produce a leather-like material.

The material was finished with a high performance coating, routinelyused in the footwear industry. The finished material was then glued overthree stripes of leather board to create a three dimensional surfacetexture and aesthetic.

EXAMPLE 19 Polyester Mesh Netting

Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd.(Medical Collagen Sponge). This source of collagen is type I collagenisolated from bovine tendon by acid treatment followed by pepsindigestion and purified by size exclusion chromatography, frozen andlyophilized. The lyophilized protein (10 grams) was dissolved in 1 litreof 0.01N HCl, pH 2 using an overhead mixer. After the collagen wasadequately dissolved, as evidenced by a lack of solid collagen sponge inthe solution (at least 1 hr mixing at 1600 rpm), 111.1 millilitres of200 millimolar sodium phosphate (pH adjusted to 11.2 with sodiumhydroxide) to raise the pH of the solution to 7.2.

The resulting collagen solution was stirred for 10 minutes and 0.1millilitres of a 20% Relugan GTW (BASF) (2% on the weight of collagen)tanning agent solution was added.

To the crosslinked collagen fibril solution was added 5 mls of 20%Tanigan FT (Lanxess) was added and stirred for one hour. FollowingTanigan-FT addition, 1 gram Expancel Microspheres (10% on the weight ofcollagen) 461 WE 20 d36 (AkzoNobel) and 40 mls (80% on the weight ofcollagen) of Truposol Ben (Trumpler) was added and added and stirred foradditional hour using an overhead stirrer. The pH of the solution waschanged to 4.0 using 10% formic acid and stirred for an hour.

After pH change three strips (each 10 mm wide) of 100% polyester meshnetting fabric was cut out and placed horizontally (with a 5 mm gap inbetween each piece) on top of a 90 mm Whatman no. 1 membrane, a thinlayer of high vacuum grease (Dow Corning) was applied around the rim ofthe membrane to hold down the material whilst filtering.

150 mL of the solution was then filtered through the textile and Whatmanno. 1 membrane using a Buchner funnel attached to a vacuum pump(pressure of −27 in Hg). Vacuum was pulled for 40 mins.

The concentrated fibril tissue was then allowed to dry in a humiditychamber at 20° C. at 65% and when it had reached 20% moisture it wasplaced in an oven at 50° C. for 2 hours. The mesh netting fabricremained integrated into the fibril tissue, resulting in a fabric-backedmaterial that had a fabric embossed pattern on its surface created bythe embedded textile.

The contraction of the concentrated fibril tissue around the meshnetting created a three-dimensional end material that self-assembled;this process can be controlled to create a desired end shape.

EXAMPLE 20 Polyester Mesh Netting

Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd.(Medical Collagen Sponge). This source of collagen is type I collagenisolated from bovine tendon by acid treatment followed by pepsindigestion and purified by size exclusion chromatography, frozen andlyophilized. The lyophilized protein (4.1 g) was dissolved in 733 mL of0.01N HCl, pH 2 using an overhead mixer.

After the collagen was adequately dissolved, as evidenced by a lack ofsolid collagen sponge in the solution (at least 1 hr mixing at 1600rpm), 82 uL of the tanning agent Relugan GTW was added to the solutionfollowed by 81 mL of a 10× PBS, pH 11.2 to raise to pH of the solutionto 7.2.

The solution was mixed for 3 min, then poured into a mold containing apiece of 100% polyester mesh netting (measuring 75 mm×200 mm) that waspinned into place—suspended 5 mm above the bottom of the mold.

The collagen solution was incubated in the silicon mold for 2 hrs at 25°C. to allow the collagen to fibrillate into a viscoelastic hydrogel,encapsulating the polyester fabric in the middle of the gel. Thefibrillated collagen hydrogel was removed from the mold and placed in700 mL of acetone in a plastic jar and shaken on an orbital shaker at 40rpm at 25° C.

The hydrogel was dehydrated by refreshing the acetone after an overnightincubation followed by 5×1 hr washes and another overnight incubation.Acetone was refreshed after each wash to remove water from the gel.Following acetone dehydration, the collagen gel was incubated inlubricating solution containing 20% (v/v) of either cod liver oil orcastor oil in 80% acetone or ethanol, respectively, overnight whileshaking at 40 rpm. Following incubation in the fat liquor solution, thecollagen gel was dried at 37° C.

After drying, the material becomes soft and leather-like. Further, theencapsulated mesh netting creates a doubled sided textured grainsurface, which can be modified almost infinitely depending on the type,and structure, of the fabric embedded.

INTERPRETATION OF DESCRIPTION

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising” means various components can be co jointlyemployed in the methods and articles (e.g., compositions and apparatusesincluding device and methods). For example, the term “comprising” willbe understood to imply the inclusion of any stated elements or steps butnot the exclusion of any other elements or steps.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “substantially”, “about” or“approximately,” even if the term does not expressly appear. The phrase“about” or “approximately” may be used when describing magnitude and/orposition to indicate that the value and/or position described is withina reasonable expected range of values and/or positions. For example, anumeric value may have a value that is +/−0.1% of the stated value (orrange of values), +/−1% of the stated value (or range of values), +/−2%of the stated value (or range of values), +/−5% of the stated value (orrange of values), +/−10% of the stated value (or range of values), etc.Any numerical range recited herein is intended to include all sub-rangessubsumed therein.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference,especially referenced is disclosure appearing in the same sentence,paragraph, page or section of the specification in which theincorporation by reference appears.

1. A composite material comprising: at least one porous, permeable, orabsorptive secondary component, and at least one biofabricated material,comprising a network of recombinant non-human collagen fibrils, whereinthe collagen contains substantially no 3-hydroxyproline; wherein saidmaterial contains no more than 25% by weight water; and wherein saidmaterial contains at least 1% of a lubricant.
 2. The composite accordingto claim 1, wherein the at least one biofabricated material has beenuniformly dispersed, encapsulated, incorporated, or otherwise introducedinto the at least one secondary material such that the concentration ofthe biofabricated material in identical unit volumes of the compositevaries by no more than 20%.
 3. The composite according to claim 1,wherein the at least one secondary material has been dispersed,encapsulated, incorporated, or otherwise introduced into a continuousphase of collagen fibrils comprised by the at least one biofabricatedmaterial such that the concentration of the secondary material inidentical unit volumes of the composite varies by no more than 20%. 4.The composite according to claim 1, which has a top and bottom surfaceor an inner and outer surface.
 5. The composite according to claim 4,wherein the biofabricated material is only on or incorporated into oneof the top, bottom, inner or outer surfaces.
 6. The composite accordingto claim 5, wherein the secondary component is a paper, fabric, or othernonwoven or woven fibrous material.
 7. The composite according to claim4, wherein the biofabricated material is on or incorporated into boththe top and bottom surfaces or both the inner or outer surfaces.
 8. Thecomposite according to claim 1, wherein the secondary componentcomprises at least one resin, polymer, or plastic.
 9. The compositeaccording to claim 1, wherein the secondary component comprises at leastone particle, bead, fiber, wire, mesh, woven, or nonwoven.
 10. Thecomposite according to claim 1, wherein the biofabricated materialcontains less than 1% by weight of actin, keratin, elastin, fibrin,albumin, globulin, mucin, mucinoids, noncollagen structural proteins,and/or noncollagen nonstructural proteins;
 11. The composite accordingto claim 1, wherein the biofabricated material is substantially free ofnon-collagenous proteins, carbohydrates, nucleic acids, or lipids, orimmunogens, antigens, or allergens found in leather.
 12. The compositeaccording to claim 1, wherein the biofabricated material comprises atleast 1% of at least one crosslinker.
 13. The composite according toclaim 1, wherein the diameters of fibrils in the biofabricated materialexhibit a substantially unimodal distribution wherein at least 70% ofthe diameters of the fibrils in the material distribute around a singlemode of diameter.
 14. The composite according to claim 1, wherein thebiofabricated material comprises at least one lubricant is selected fromthe group consisting of at least one fat, biological, mineral orsynthetic oil, sulfonated oil, polymer, and organofunctional siloxane.15. The composite of claim 1, wherein the biofabricated materialcomprises at least one photoluminescent agent.
 16. The compositeaccording to claim 1, wherein the biofabricated material comprises atleast one electrically conductive or magnetic agent.
 17. The compositeaccording to claim 1, wherein the biofabricated material has an elasticmodulus between 100 kPa and 100 MPa, wherein the elastic modulus variesby no more than 20% when measured at right angles across identicallengths of the material and that has a tensile strength of ranging from1 MPa to 100 MPa, wherein the tensile strength varies by no more than20% when measured at right angles across identical lengths of thematerial.
 18. The composite according to claim 1, wherein thebiofabricated material further comprises a surface coating or surfacefinish; wherein the surface coating or surface finish is distributeduniformly throughout the material such that its concentration by weightin or on identical unit volumes of the material varies by no more than20%.
 19. The composite according to claim 1, wherein the biofabricatedmaterial further comprises a dye, stain, resin, polymer, pigment orpaint, wherein the dye, stain, resin, pigment or paint is distributeduniformly throughout the material such that its concentration by weightin or on identical unit volumes of the material varies by no more than20%.
 20. The composite according to claim 1, wherein the biofabricatedmaterial further comprises at least one filler, wherein the filler isdistributed uniformly throughout the material such that itsconcentration by weight in or on identical unit volumes of the materialvaries by no more than 20%.